Genotypic Characterization of Rickettsia bellii Reveals...
9
Research Article Genotypic Characterization of Rickettsia bellii Reveals Distinct Lineages in the United States and South America Felipe S. Krawczak , 1 Marcelo B. Labruna , 1 Joy A. Hecht , 2 Christopher D. Paddock , 2 and Sandor E. Karpathy 2 1 Department of Preventive Veterinary Medicine and Animal Health, Faculty of Veterinary Medicine, University of S˜ ao Paulo, S˜ ao Paulo, SP, Brazil 2 Rickettsial Zoonoses Branch, National Center for Emerging and Zoonotic Infectious Diseases, Centers for Disease Control and Prevention, Atlanta, GA, USA Correspondence should be addressed to Sandor E. Karpathy; [email protected] Received 17 November 2017; Revised 11 February 2018; Accepted 28 February 2018; Published 8 April 2018 Academic Editor: Stephan Koblm¨ uller Copyright © 2018 Felipe S. Krawczak et al. is is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. e bacterium Rickettsia bellii belongs to a basal group of rickettsiae that diverged prior to the pathogenic spotted fever group and typhus group Rickettsia species. Despite a diverse representation of R. bellii across more than 25 species of hard and soſt ticks in the American continent, phylogeographical relationships among strains of this basal group-Rickettsia species are unknown; the work described here explores these relationships. DNA was extracted from 30 R. bellii tick isolates: 15 from the United States, 14 from Brazil, and 1 from Argentina. A total of 2,269 aligned nucleotide sites of 3 protein coding genes (gltA, atpA, and coxA) and 2 intergenic regions (rpmE-tRNA fmet and RC1027-xthA2) were concatenated and subjected to phylogenetic analysis by Bayesian methods. Results showed a separation of almost all isolates between North and South Americas, suggesting that they have radiated within their respective continents. Phylogenetic positions of the 30 isolates could be a result of not only their geographical origin but also the tick hosts they have coevolved with. Whether R. bellii originated with ticks in North or South America remains obscure, as our analyses did not show evidence for greater genetic divergence of R. bellii in either continent. 1. Introduction Members of the genus Rickettsia (Rickettsiales: Rickettsi- aceae) are Gram-negative obligate intracellular bacteria, usu- ally in association with arthropods, although a number of distinct genotypes have also been described from leeches, amoeba, ciliate, and hydra [1]. Phylogenetic analysis-based studies have classified the Rickettsia species into five major groups, namely, the spotted fever group (SFG), the typhus group (TG), the transitional group (TRG), the Rickettsia canadensis group, and the Rickettsia bellii group [1, 2]. is later group occupies a basal position in all major phylogenetic studies, which indicates early divergence within the genus [1, 3, 4]. Rickettsia bellii was formally described in 1983 based on isolates obtained from multiple species of ixodids (hard ticks) and argasids (soſt ticks) in the United States [5]. In the United States, R. bellii has been reported to be infecting the following 8 tick species: Dermacentor variabilis, Dermacentor occidentalis, Dermacentor andersoni, Dermacentor albipictus, Dermacentor parumapertus, Haemaphysalis leporispalustris, Ornithodoros concanensis, and Argas cooleyi [5]. In recent years, investigators in Latin America have identified R. bellii in ixodid ticks throughout Latin America, including El Salvador, Costa Rica, Panama, Colombia, Brazil, Peru, and Argentina [6–13]. In Central and South America, R. bellii has been detected in at least 19 tick species, namely, Ixodes loricatus, Haemaphysalis juxtakochi, Amblyomma aureolatum, Amblyomma dubitatum, Amblyomma humerale, Amblyomma incisum, Amblyomma neumanni, Amblyomma longirostre, Amblyomma naponense, Amblyomma nodosum, Hindawi BioMed Research International Volume 2018, Article ID 8505483, 8 pages https://doi.org/10.1155/2018/8505483
Transcript of Genotypic Characterization of Rickettsia bellii Reveals...
Research ArticleGenotypic Characterization of Rickettsia bellii Reveals DistinctLineages in the United States and South America
Felipe S Krawczak 1 Marcelo B Labruna 1 Joy A Hecht 2
Christopher D Paddock 2 and Sandor E Karpathy 2
1Department of Preventive Veterinary Medicine and Animal Health Faculty of Veterinary MedicineUniversity of Sao Paulo Sao Paulo SP Brazil2Rickettsial Zoonoses Branch National Center for Emerging and Zoonotic Infectious DiseasesCenters for Disease Control and Prevention Atlanta GA USA
Correspondence should be addressed to Sandor E Karpathy skarpathycdcgov
Received 17 November 2017 Revised 11 February 2018 Accepted 28 February 2018 Published 8 April 2018
Academic Editor Stephan Koblmuller
Copyright copy 2018 Felipe S Krawczak et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited
The bacterium Rickettsia bellii belongs to a basal group of rickettsiae that diverged prior to the pathogenic spotted fever group andtyphus group Rickettsia species Despite a diverse representation of R bellii across more than 25 species of hard and soft ticks inthe American continent phylogeographical relationships among strains of this basal group-Rickettsia species are unknown thework described here explores these relationships DNA was extracted from 30 R bellii tick isolates 15 from the United States 14from Brazil and 1 from Argentina A total of 2269 aligned nucleotide sites of 3 protein coding genes (gltA atpA and coxA) and2 intergenic regions (rpmE-tRNAfmet and RC1027-xthA2) were concatenated and subjected to phylogenetic analysis by Bayesianmethods Results showed a separation of almost all isolates between North and South Americas suggesting that they have radiatedwithin their respective continents Phylogenetic positions of the 30 isolates could be a result of not only their geographical originbut also the tick hosts they have coevolved withWhetherR bellii originated with ticks in North or South America remains obscureas our analyses did not show evidence for greater genetic divergence of R bellii in either continent
1 Introduction
Members of the genus Rickettsia (Rickettsiales Rickettsi-aceae) are Gram-negative obligate intracellular bacteria usu-ally in association with arthropods although a number ofdistinct genotypes have also been described from leechesamoeba ciliate and hydra [1] Phylogenetic analysis-basedstudies have classified the Rickettsia species into five majorgroups namely the spotted fever group (SFG) the typhusgroup (TG) the transitional group (TRG) the Rickettsiacanadensis group and the Rickettsia bellii group [1 2] Thislater group occupies a basal position in allmajor phylogeneticstudies which indicates early divergence within the genus[1 3 4]
Rickettsia bellii was formally described in 1983 based onisolates obtained frommultiple species of ixodids (hard ticks)
and argasids (soft ticks) in the United States [5] In theUnited States R bellii has been reported to be infecting thefollowing 8 tick speciesDermacentor variabilis Dermacentoroccidentalis Dermacentor andersoni Dermacentor albipictusDermacentor parumapertus Haemaphysalis leporispalustrisOrnithodoros concanensis and Argas cooleyi [5] In recentyears investigators in Latin America have identified Rbellii in ixodid ticks throughout Latin America includingEl Salvador Costa Rica Panama Colombia Brazil Peruand Argentina [6ndash13] In Central and South America Rbellii has been detected in at least 19 tick species namelyIxodes loricatus Haemaphysalis juxtakochi Amblyommaaureolatum Amblyomma dubitatum Amblyomma humeraleAmblyomma incisum Amblyomma neumanni Amblyommalongirostre Amblyomma naponense Amblyomma nodosum
HindawiBioMed Research InternationalVolume 2018 Article ID 8505483 8 pageshttpsdoiorg10115520188505483
2 BioMed Research International
Amblyomma ovale Amblyomma oblongoguttatum Ambly-omma parvum Amblyomma pseudoconcolor Amblyommarotundatum Amblyomma sabanerae Amblyomma scalptura-tum Amblyomma tigrinum andAmblyomma varium [6 7 1214ndash16]
The wide host range and extraordinarily broad distribu-tion of this Rickettsia species are intriguing and although Rbellii is generally considered as nonpathogenic for animalsand humans [2 17] it could play an important role in theecology and epidemiology of other pathogenic tick-borneSFG rickettsiae in the Americas [18] Despite the diverserepresentation of R bellii across more than 25 species of hardand soft ticks in the American continent the phylogeograph-ical relationships of this basal group-Rickettsia species hasnot been examined the work described here explores theserelationships based on a hypothesis that R bellii could haveevolved firstly in one continent or a particular tick group andthen radiated to other continents or other tick groups
2 Materials and Methods
21 Rickettsial Isolates Rickettsia bellii was isolated fromvarious species of hard ticks collected in South America andNorth America as described in the original publication ofeach of the isolates listed in Table 1 A total of 30 isolates of Rbellii from 13 species of hard ticks from 3 countries were usedfor the analysis (Figure 1) These included 1 from Argentina14 from Brazil and 15 from USA (Table 1) The Brazilian andArgentinean isolates are available at the Rickettsial Collectionof the Laboratory of Parasitic Diseases of Faculdade deMedicina Veterinaria e Zootecnia (FMVZ) of the Universityof Sao Paulo (USP) and the US isolates are available atthe Rickettsial Zoonoses Branch at the Centers for DiseaseControl and Prevention (CDC) Atlanta Georgia USA
22 DNAExtraction and PCR DNA from theR bellii isolatesfrom South America was extracted using the DNeasy Bloodand Tissue Kit (Qiagen Valencia CA) and the QIAampDNAMini Kit (Qiagen) was used for the 15 isolates from NorthAmerica all in accordance with the manufacturerrsquos recom-mendations Amplification of fragments of five rickettsialgenes and fifteen intergenic regions was attempted with theprimer pairs listed in Table 2 Each PCR reaction consistedof 2120583l template DNA 20 picomoles of each primer and10 120583l Taq PCRMaster Mix (Qiagen) while cycling conditionsincluded a 1-minute incubation at 95∘C followed by 35 cyclesof a 30-second denaturation at 95∘C a 30-second annealingincubation (Table 2) and a 1-minute extension at 72∘C Thiswas followed by a final 10-minute extension at 72∘C GradientPCR was used to optimize the annealing temperatures in Rbellii for each primer pair
23 DNA Purification and Sequencing DNA fragments am-plified by PCR were visualized using a UV lamp in a 15agarose gel containing 01120583gml ethidium bromide PCRproducts of the appropriate size were cut from the gel andthen purified using the Wizard SV gel and PCR clean-upsystem (Promega Madison WI) Sequencing reactions wereprepared using one microliter of purified PCR product and
the BigDye Terminator v31 Cycle Sequencing Kit (AppliedBiosystems Foster City CA) according to the manufacturerrsquosinstructions and then sequenced using an ABI 3100 geneticanalyzer (Applied Biosystems) Each PCR amplicon wassequenced at least once in both directions
24 Sequence Alignment and Phylogenetic Inferences Theresulting sequences were assembled using Geneious 914software (Biomatters Ltd Auckland New Zealand) andaligned using ClustalW [35] and MEGA 606 software [36]The resulting alignment was examined by eye to ensureproper alignment of the sequences and the simple indel-coding method [37] was used to remove insertionsdeletionsMrBayes 326 [38] was used in Geneious to perform aBayesian phylogenetic analysis The Jukes-Cantor model wasused in an analysis consisting of 10000000 generations(1000000 burn-ins) The analysis included 3 heated chainsand an effective sample size (ESS) of 901 was achieved for allparameters
25 AccessionNumbers TheGenBank accession numbers forthe DNA sequences generated in this study for the 30 Rbellii isolates shown in Table 1 are the following gltA gene(MF154866ndashMF154895) atpA gene (MF154926ndashMF154955)coxA gene (MF154896ndashMF154925) rpmE-tRNAfmet inter-genic region (MF154956ndashMF154985) and RC1027-xthA2intergenic region (MF154986ndashMF155015)
3 Results
Among the 20 primer pairs used for amplification of rick-ettsial DNA fragments only the primer pairs 15 16 1719 and 20 (Table 2) were successful in amplifying DNAfrom the R bellii isolates These primers corresponded to 3protein coding genes (gltA atpA and coxA) and 2 intergenicregions (rpmE-tRNAfmet and RC1027-xthA2)The nucleotidesequences from the five loci for each of the 30 isolates wereconcatenated and used to perform a Bayesian analysis oftheir phylogeny There was a separation of the isolates intoat least three clades one representing all South Americanisolates and the United States isolate CA-459 cultivated froma H leporispalustris tick collected in northern California[39] one comprising 6 isolates from D variabilis collectedin northern California [29] and a third clade comprising 5isolates from D variabilis collected in Ohio [30] (Figure 1)North American isolates from D parumapertus and the 369-C strain from D variabilis did not group within any of theabove clades Within the South American clade there weretwo smaller well-supported clades (each with a posteriorprobability of 10) one consisting of three isolates associatedwith I loricatus ticks and one consisting of two A ovale-associated isolates
Overall there were very few polymorphisms (lt05)between the 30 R bellii isolates In the 2269-nucleotidealignment identity values between the isolates varied from9956 to 100 (Table S1) DNA sequences of the isolatescomposing the main South American clade were 9969ndash100identical and at the same time 9956ndash9974 identical to theclade containing D variabilis isolates from California and
BioMed Research International 3
Minas Gerais 2 9
Rio Grande doSul 10 11
Mato Grosso do Sul 13
Ohio 23 25ndash27 29
California 17ndash22 30
Arkansas 16Utah 28
Texas 24
United States
Brazil
Argentina
IL-Mogi Ixodes loricatus (8)IL-RS1 Ixodes loricatus (10) RB-CL Ixodes loricatus (11)
AO Amblyomma ovale (6)P-10 Amblyomma ovale (14)
Mogi Amblyomma aureolatum (1) Guarda-Mor Amblyomma dubitatum (2) Ad-CORD-SP Amblyomma dubitatum (3) Ad-25-INT Amblyomma dubitatum (4) PNSM Amblyomma incisum (5)
HJ4 Haemaphysalis juxtakochi (7)
Ap GSV 136 Amblyomma parvum (9)HJ1 Haemaphysalis juxtakochi (12)
Rb MS PSEUDO Amblyomma pseudoconcolor (13) An4 Amblyomma neumanni (15)
CA-459 Haemaphysalis leporispalustris (30)
CA13-1 Dermacentor variabilis (17)CA13-9 Dermacentor variabilis (18)CA13-17 Dermacentor variabilis (19)Putah Creek Dermacentor variabilis (20)Yolo Dermacentor variabilis (21)Stevenson Bridge Dermacentor variabilis (22)
OSU 83-1223 Dermacentor variabilis (23) OSU 83-117 Dermacentor variabilis (25)OSU 85-1299 Dermacentor variabilis (26)OSU 83-452 Dermacentor variabilis (27) OSU 85-389 Dermacentor variabilis (29)
TX15-1 Dermacentor parumapertus (24)369C Dermacentor variabilis (16)
Skull Valley Dermacentor parumapertus (28)
1 3ndash8 12 14 So PauloCＩrdoba 15
20E minus 4
059
074
1
1
1
Figure 1 Molecular phylogenetic analysis of 30 isolates of Rickettsia bellii from North and South America
9974ndash9991 identical to the clade containing D variabilisisolates from Ohio Within-clade identities were 9974ndash100for the D variabilis isolates from Ohio and 100 for the Dvariabilis isolates from California the Ohio clade was 9974identical to the California clade Finally sequences of thethree North American isolates (two from D parumapertusand one from D variabilis) that did not group within anymain clade were 9987ndash9996 identical to each other and9960ndash9991 identical to the remaining isolates
4 Discussion
Here we performed for the first time a phylogenetic anal-ysis of multiple isolates of R bellii Because these isolatesrepresented a number of different locations in North andSouth America our intention was to infer phylogeographicalrelationships Indeed there was a separation of almost allisolates between the two continents suggesting that they haveradiated within their respective continents Although theposterior probability of the separation of the South Americanclade from the North American isolates was not strong (PP =059) this topology was also achieved using both the GTRand HKY85 evolutionary models (supplemental material)giving additional evidence for this separation At the sametime it is noteworthy that while the North American cladesrepresented isolates exclusively from Dermacentor ticks theSouth American clade was composed of isolates from thegenera Amblyomma Haemaphysalis and Ixodes Under suchcircumstances the phylogenetic positions of the 30 isolatesof the present study could be a result of not only theirgeographical origin but also the tick hosts in which theywere isolated from This later assumption is corroborated by
the position of the isolate from H leporispalustris whichdespite being from North America grouped within theSouth American clade where at least two other isolates fromHaemaphysalis ticks were present
To our knowledge the number and diversity of tickspecies infected with R bellii are the largest and broad-est described among species in the genus Rickettsia Themagnitude of different host species indicates horizontaltransmission among tick populations especially in SouthAmerica where isolates fromdifferent tick species and generagrouped together in the same clade It is generally acceptedthat R bellii is not pathogenic for vertebrates in fact Rbellii has never been isolated from a vertebrate host [2]On the other hand there has been serological evidence ofanimal natural infection or exposure by R bellii [40 41]While R bellii horizontal transmission among ticks via ver-tebrate host cannot be discarded another likely mechanismcould be via tick parasitoids since the parasitism of thehymenopteran Ixodiphagus spp is relatively common amongdifferent tick genera in the Americas [42 43] In fact a recentstudy provided molecular detection of tick-borne rickettsiaein Ixodiphagus wasps that had emerged from ticks [44]highlighting the possibility that rickettsial organisms couldbe shared by ticks and their parasitoids Regardless of themechanisms of horizontal transmission the tendency of eachR bellii genotype to be associated with a different tick species(Figure 1) suggests that horizontal transmission was moreefficient at earlier times thereaftermost of theR bellii isolatesare likely to have coevolved specifically with their specific tickspecies host possibly towards a symbiotic association In factanalysis of the genome of the type strain of R bellii has shownfeatures compatible with various symbiotic bacteria such as
4 BioMed Research International
Table 1 Rickettsia bellii isolates from South and North America used in the present study
Numberlowast Isolate name Tick host Geographic origin Reference(1) Mogi Amblyomma aureolatum Mogi das Cruzes-SP Brazil [19](2) Ad-MG Amblyomma dubitatum Guarda-Mor-MG Brazil [16](3) Ad-CORD-SP A dubitatum Cordeiropolis -SP Brazil [20](4) Ad-25-INT A dubitatum Ribeirao Grande-SP Brazil [20](5) PNSM Amblyomma incisum Cubatao-SP Brazil [21](6) AO Amblyomma ovale Ribeirao Grande-SP Brazil [22](7) HJ4 Haemaphysalis juxtakochi Ribeirao Grande-SP Brazil [23](8) IL-Mogi Ixodes loricatus Mogi das Cruzes-SP Brazil [24](9) Ap GSV 136 Amblyomma parvum Chapada Gaucha-MG Brazil [25](10) IL-RS1 I loricatus Derrubadas-RS Brazil [26](11) RB-CL I loricatus Cerro Largo-RS Brazil [13](12) HJ1 H juxtakochi Sao Paulo-SP Brazil [23](13) Ap-MS Amblyomma pseudoconcolor Corumba-MS Brazil [16](14) P-10 A ovale Peruıbe-SP Brazil [27](15) An4 Amblyomma neumanni Dean Funes-Cordoba Argentina [28](16) 369-C Dermacentor variabilis Washington Co Arkansas USA [5](17) CA13-1 Dermacentor variabilis Yolo Co California USA [29](18) CA13-9 D variabilis Yolo Co California USA [29](19) CA13-17 D variabilis Yolo Co California USA [29](20) Putah Creek D variabilis Solano Co California USA [29](21) Yolo D variabilis Yolo Co California USA [29](22) Stevenson Bridge D variabilis Yolo Co California USA [29](23) OSU 83-1223 D variabilis Knox County Ohio USA [30](24) TX15-1 Dermacentor parumapertus Brewster County Texas USA [31](25) OSU 83-117 D variabilis Licking County Ohio USA [30](26) OSU 85-1299 D variabilis Morrow County Ohio USA [30](27) OSU 83-452 D variabilis Coshocton County Ohio USA [30](28) Skull Valley D parumapertus Tooele County Utah USA [31](29) OSU 85-389 D variabilis Ohio USA [30](30) CA-459 Haemaphysalis leporispalustris Mendocino Co California USA [32]lowastThese numbers are represented in Figure 1 as the geographical location of each isolate
a large genome size with high coding capacity in contrastto the reduced genome size with low coding capacity of thepathogenic rickettsiae [4 45]
It has been proposed that the R bellii group divergedprior to the division between the SFG and the TG forming abasal group that also includes various herbivorous arthropodsymbionts [1 46] While the genus Rickettsia was consideredto be approximately 150 million years old the divergenceof the R bellii group was estimated to have occurred muchmore recently around 50 million years ago [1] Analysesof the oldest tick fossils from the Cretaceous indicate thatextant tick genera (Amblyomma Ornithodoros)were soundlyestablished around 100 million years ago [47ndash49] Fromthese data it can be inferred that R bellii likely radiatedwith its principal hosts approximately 50 million years agoWhile ticks and rickettsiae are distributed in all continentsit is noteworthy that under natural conditions and withouthuman interference tick species from the NewWorld do notoccur in the Old World and vice versa the only exceptionsare a few tick species that are associated with transcontinental
marine birds [50] Indeed this scenario has accounted forthe presence of R bellii-infected ticks restricted to the NewWorld regardless of the ability of this bacterium to infecta vast array of tick genera and species On the other handwhether R bellii originated with ticks in North or SouthAmerica remains obscure since our phylogenetic analysesdid not show any evidence for greater genetic divergenceof R bellii in any of the two continents Further analysesencompassing more R bellii isolates from different tickgenera and species encompassing more molecular markersshould provide cues for the origin and radiation of R bellii inthe NewWorld
5 Conclusion
Phylogeographical analysis of 30 strains (15 from NorthAmerica and 15 from South America) isolated from 13species of 4 genera shows a clear differentiation betweenmost of the North and South American isolates indicatinggeographic isolation between isolates of these two continents
BioMed Research International 5
Table2Prim
erpairs
used
fora
mplificatio
nof
rickettsialgenes
orintergenicregion
sinthep
resent
study
Prim
erpair
Target
Forw
ardprim
er(51015840to
31015840)
Reversep
rimer
(51015840to
31015840)
Ann
ealin
gtemperature
(∘ C)
Amplicon
size(nt)
Reference
(1)
mppA-purC
GCA
ATTA
TCGGTC
CGAAT
GTT
TCAT
TTAT
TTGTC
TCAAAAT
TCA
45to
551
160
[33]
(2)
nusG
-rplK
CAGTT
GCA
ATAT
TGGTA
AAG
CACA
GCA
GCT
GGAAT
TATC
AAG
TT45
to551
270
[33]
(3)
murG-RC0
563
GAAG
AAAAG
AAG
GGCA
TAAG
CTA
CAAG
CTGAAAG
TAAAAAC
ATTC
C40
to521
293
[33]
(4)
ntrY-rpsU
AGCT
GCT
GTT
GCT
AAAG
TAAAAA
CAAG
AAG
CAGCA
AGAAG
ACAG
A52
to641
363
[33]
(5)
dksA-xerC
TCCC
ATAG
GTA
ATTT
AGGTG
TTTC
TACT
ACCG
CATA
TCCA
ATTA
AAAA
45to
551
416
[33]
(6)
spo0J-a
bcT1
AAAG
ATTT
GGAAG
AAT
TAGAC
TTGAT
TTTG
CTTA
AAC
CAAC
CATT
TCA
45to
551
259
[33]
(7)
fabZ
-lpxD
TGTT
AGGAT
CGAT
TTTA
AGTA
CTCT
ATCT
TGGAT
TGGCA
TAGAC
AAT
CTAT
TA45
to551
195
[33]
(8)
RC1137-tlc-5
CGGGAT
AAC
GCC
GAG
TAAT
AAT
GCC
GCT
CTGAAT
TTGTT
T45
to551
264
[33]
(9)
RC0230-RC0
231
TGCA
CCCG
CCTA
AAAC
TAAC
ATGGTC
GGCC
GTA
GAAAAA
45to
551
232
[33]
(10)
groE
S-RC
0970
CTTG
CATC
GGCT
TTTC
TTTT
AGCT
TTGAG
CTGAT
GGGCT
A45
to551
215
[33]
(11)
rrf-p
yrH
GAG
CTTT
CTCC
ATCT
TTTC
TTG
AAAG
GGGAAT
ATAC
GAC
AAT
TGAG
45to
551
238
[33]
(12)
tRNA
Gly-tR
NA
Tyr
AGCT
TGGAAG
GCT
GGAAC
TCAT
CCTT
CTCC
CTCC
ACCA
CT45
to551
148
[33]
(13)
pal-R
C1201
TGCA
AGCA
CACA
TAAT
GCA
ATC
AAAAT
CGAT
TCCT
CTTT
TCC
45to
551
216
[33]
(14)
atpA
ATCA
AGCG
TTGCA
CAGAT
AGGGAAG
TGCC
GTA
AGTG
AAC
C58
Unk
nownlowast
[1]
(15)
rpmE-tRNA
fMet
TTCC
GGAAAT
GTA
GTA
AAT
CAAT
CTC
AGGTT
ATGAG
CCTG
ACGA
54144
[33]
(16)
RC1027-xthA2
GGTA
TGTA
AAT
GAG
CCTT
ATCA
ATAC
TTC
AGTA
GTA
TAAG
TAGCT
CCTG
CTGTC
54351
[33]
(17)
gltA
GTC
TACT
GCT
TCGTG
TAGAT
CAAC
GGCT
GAC
CTAT
AGAAT
ATTT
ATAAG
AC54
408
[25]
(18)
coxA
ACAG
CCGTT
GAT
ATGGCT
ACA
TATT
CCAAC
CGGCA
AAAG
58Unk
nownlowast
[1]
(19)
coxA
GGTG
CTCC
TGAT
ATGGCA
TTCA
TATT
CCAG
CCGGCA
AAAG
58Unk
nownlowast
[1]
(20)
atpA
ATCA
AGCG
TTGCA
CAGAT
AGCG
ACTT
ACCG
AAAT
ACCG
AC56
449
[134]
lowastDatan
otgivenin
theo
riginalpu
blication1teste
din
gradient
PCR
6 BioMed Research International
Additionally this analysis separated isolates of R bellii by thespecies of tick fromwhich these were isolated indicating thatthe isolates could have coevolved with their tick vectors overtime
Disclosure
The findings and conclusions are those of the authors anddo not necessarily reflect the views of the US Departmentof Health and Human Services or the official position of theCenters for Disease Control and Prevention
Conflicts of Interest
The authors declare that there are no conflicts of interestregarding the publication of this manuscript
Acknowledgments
This work received financial support from the Sao PauloResearch Foundation (FAPESP Grant 201510060-6)
Supplementary Materials
An identity matrix table of the 2269-nucleotide alignmentused for the phylogeny if R bellii is available in thesupplemental material for this article (Table S1) The finalalignment comprising 2269 nucleotides was concatenated inthe following order rpmE -tRNAfmet (414-nt) gltA (357-nt) coxA (898-nt) atpA (449-nt) and RC1027 -xthA2 (151-nt) and it is also available in the supplementary material(Rbellii final alignmentfas) Two additional phylogenetictrees are also available in the supplementary material (RbelliiGTR treepdf and Rbellii HKY treepdf) Table S1 identitymatrix of a 2269-nucleotide alignment of the 30 Rickettsiabellii isolates from Brazil (BRA) Argentina (ARG) and theUnited States (USA) used in the present study SupplementaryFigure 1 molecular phylogenetic analysis of 30 isolates ofRickettsia bellii from North and South America A total of2269 aligned nucleotide sites of 3 protein coding genes (gltAatpA and coxA) and 2 intergenic regions ( rpmE-tRNAfmet and RC1027- xthA2) were concatenated and subjectedto Bayesian analysis A total of 10000000 generations wererun using the GTRmodel with a sample frequency of 10000The analysis was run with 3 heated chains and the first1000000 generations were discarded as burn-in Numbers atnodes are support values derived from posterior probabilityThe scale bar is in units of expected substitutions per siteSupplementary Figure 2 molecular phylogenetic analysis of30 isolates of Rickettsia bellii fromNorth and South AmericaA total of 2269 aligned nucleotide sites of 3 protein codinggenes (gltA atpA and coxA) and 2 intergenic regions(rpmE-tRNA fmet and RC1027- xthA2) were concatenatedand subjected to Bayesian analysis A total of 10000000generations were run using the HKY85 model with a samplefrequency of 10000 The analysis was run with 3 heatedchains and the first 1000000 generations were discarded asburn-in Numbers at nodes are support values derived from
posterior probability The scale bar is in units of expectedsubstitutions per site (Supplementary Materials)
References
[1] L A Weinert J H Werren A Aebi G N Stone and F MJiggins ldquoEvolution and diversity of Rickettsia bacteriardquo BMCBiology vol 7 article no 6 2009
[2] P Parola C D Paddock C Socolovschi et al ldquoUpdate on tick-borne rickettsioses around the world a geographic approachrdquoClinical Microbiology Reviews vol 26 no 4 pp 657ndash702 2013
[3] D R Stothard J B Clark and P A Fuerst ldquoAncestral diver-gence of Rickettsia bellii from the spotted fever and typhusgroups of Rickettsia and antiquity of the genus Rickettsiardquo Inter-national Journal of Systematic Bacteriology vol 44 no 4 pp798ndash804 1994
[4] V Merhej and D Raoult ldquoRickettsial evolution in the light ofcomparative genomicsrdquo Biological Reviews vol 86 no 2 pp379ndash405 2011
[5] R N Philip E A Casper and R L Anacker ldquoRickettsia belliisp nov A tick-borne Rickettsia widely distributed in theUnited States that is distinct from the spotted fever and typhusbiogroupsrdquo International Journal of Systematic Bacteriology vol33 no 1 pp 94ndash106 1983
[6] M B Labruna S Mattar V and S Nava ldquoRickettsioses inLatin America Caribbean Spain and Portugalrdquo Revista MVZCordoba vol 16 no 2 pp 2435ndash2457 2011
[7] M Ogrzewalska I Literak J M Cardenas-CallirgosM Capekand M B Labruna ldquoRickettsia bellii in ticks Amblyommavarium Koch 1844 from birds in Perurdquo Ticks and Tick-borneDiseases vol 3 no 4 pp 254ndash256 2012
[8] M Ogrzewalska D G Saraiva J Moraes-Filho et al ldquoEpidemi-ology of Brazilian spotted fever in the Atlantic Forest state ofSao Paulo Brazilrdquo Parasitology vol 139 no 10 pp 1283ndash13002012
[9] J Miranda and S Mattar ldquoMolecular detection of Rickettsiabellii and Rickettsia sp strain Colombianensi in ticks fromCordoba Colombiardquo Ticks and Tick-borne Diseases vol 5 no2 pp 208ndash212 2014
[10] M Andoh A Sakata A Takano et al ldquoDetection of Rickettsiaand Ehrlichia spp In ticks associated with exotic reptiles andamphibians imported into Japanrdquo PLoS ONE vol 10 no 7Article ID e0133700 2015
[11] M Ogrzewalska I Literak M Capek et al ldquoBacteria of thegenus Rickettsia in ticks (Acari Ixodidae) collected from birdsin Costa Ricardquo Ticks and Tick-borne Diseases vol 6 no 4 pp478ndash482 2015
[12] A R M Barbieri L Romero and M B Labruna ldquoRickettsiabellii infecting Amblyomma sabanerae ticks in El SalvadorrdquoPathogens and Global Health vol 106 no 3 pp 188-189 2012
[13] F S Krawczak L C Binder C S Oliveira et al ldquoEcology of atick-borne spotted fever in southern Brazilrdquo Experimental andApplied Acarology vol 70 no 2 pp 219ndash229 2016
[14] D McIntosh R A Bezerra H R Luz et al ldquoDetectionof Rickettsia bellii and Rickettsia amblyommii in Amblyommalongirostre (Acari Ixodidae) fromBahia state Northeast BrazilrdquoBrazilian Journal of Microbiology vol 46 no 3 pp 879ndash8832015
[15] H S Soares A R M Barbieri T F Martins et al ldquoTicks andrickettsial infection in thewildlife of two regions of the BrazilianAmazonrdquo Experimental and Applied Acarology vol 65 no 1 pp125ndash140 2015
BioMed Research International 7
[16] F B Costa A R M Barbieri M P J Szabo V N Ramos UPiovezan and M B Labruna ldquoNew records of Rickettsia bellii-infected ticks in Brazilrdquo Brazilian Journal of Veterinary Researchand Animal Science vol 54 no 1 pp 92ndash95 2017
[17] M B Labruna T Whitworth M C Horta et al ldquoRickettsiaspecies infecting Amblyomma cooperi ticks from an area inthe state of Sao Paulo Brazil where Brazilian Spotted Fever isendemicrdquo Journal of Clinical Microbiology vol 42 no 1 pp 90ndash98 2004
[18] K R Macaluso D E Sonenshine S M Ceraul and A F AzadldquoRickettsial infection in Dermacentor variabilis (Acari Ixodi-dae) inhibits transovarial transmission of a second RickettsiardquoJournal of Medical Entomology vol 39 no 6 pp 809ndash813 2002
[19] A Pinter and M B Labruna ldquoIsolation of Rickettsia rickettsiiand Rickettsia bellii in cell culture from the tick Amblyommaaureolatum in Brazilrdquo Annals of the New York Academy ofSciences vol 1078 pp 523ndash529 2006
[20] R De Campos Pacheco M C Horta A Pinter et al ldquoSurvey ofRickettsia spp in the ticks Amblyomma cajennense and Ambly-omma dubitatum in the State of Sao Paulordquo Journal of theBrazilian Society of Tropical Medicine vol 42 no 3 pp 351ndash3532009
[21] G S Sabatini Pesquisa de Carrapatos e Riquetsias no ParqueEstadual da Serra do Mar Nucleo Itutinga ndash Pilloes Sao Paulo[Dissertation thesis] University of Sao Paulo Sao Paulo SP2010
[22] R Pacheco S Rosa L Richtzenhain M P J Szabo and M BLabruna ldquoIsolation of Rickettsia bellii from Amblyomma ovaleand Amblyomma incisum ticks from southern Brazilrdquo RevistaMVZ Cordoba vol 13 no 2 pp 1273ndash1279 2008
[23] M B Labruna R C Pacheco L J Richtzenhain and M P JSzabo ldquoIsolation of Rickettsia rhipicephali and Rickettsia belliifrom Haemaphysalis juxtakochi ticks in the state of Sao PauloBrazilrdquo Applied and Environmental Microbiology vol 73 no 3pp 869ndash873 2007
[24] M C Horta A Pinter T T S Schumaker and M B LabrunaldquoNatural infection transovarial transmission and transstadialsurvival of Rickettsia bellii in the tick Ixodes loricatus (AcariIxodidae) from Brazilrdquo Annals of the New York Academy ofSciences vol 1078 pp 285ndash290 2006
[25] A R M Barbieri Ecologia de carrapatos e rickettsias transmiti-das por carrapatos em uma reserva natural de Cerrado brasileiro[PhD thesis] University of Sao Paulo Sao Paulo SP 2016
[26] F S KrawczakPhD thesis University of Sao Paulo Sao Paulo SPPesquisa de infeccao por riquetsias do grupo da febre maculosaem caes pequenos mamıferos e carrapatos em area endemica enao endemicas nos biomas Pampa eMata Atlantica no estado doRio Grande do Sul [PhD thesis] University of Sao Paulo SaoPaulo SP 2016
[27] M P J Szabo F A Nieri-BastosM G Spolidorio T FMartinsA M Barbieri and M B Labruna ldquoIn vitro isolation fromAmblyomma ovale (Acari Ixodidae) and ecological aspects ofthe Atlantic rainforest Rickettsia the causative agent of a novelspotted fever rickettsiosis in Brazilrdquo Parasitology vol 140 no 6pp 719ndash728 2013
[28] M B Labruna R C Pacheco S Nava P E Brandao L JRichtzenhain and A A Guglielmone ldquoInfection by Rickettsiabellii and Candidatus ldquoRickettsia amblyommiirdquo in Amblyommaneumanni ticks from ArgentinardquoMicrobial Ecology vol 54 no1 pp 126ndash133 2007
[29] J A Hecht M E J Allerdice F S Krawczak M B Labruna CD Paddock and S E Karpathy ldquoDevelopment of a Rickettsia
bellii-specific TaqMan assay targeting the citrate synthase generdquoJournal of Medical Entomology vol 53 no 6 pp 1492ndash14952016
[30] K Poetter PhD thesis Molecular population and evolutionarygenetics of Rickettsia Ohio State University Columbus OHUSA 1989
[31] C D Paddock M E J Allerdice S E Karpathy et al ldquoUniquestrain of Rickettsia parkeri associated with the hard tickDerma-centor parumapertus Neumann in the western United StatesrdquoApplied and Environmental Microbiology vol 83 no 9 ArticleID e03463-16 2017
[32] R N Philip E A Casper and R S Lane ldquoSerotypes of tick-borne spotted fever group Rickettsiae from western CaliforniardquoTheAmerican Journal of Tropical Medicine and Hygiene vol 30no 3 pp 722ndash727 1981
[33] P-E Fournier Y Zhu H Ogata and D Raoult ldquoUse of highlyvariable intergenic spacer sequences for multispacer typing ofRickettsia conorii strainsrdquo Journal of Clinical Microbiology vol42 no 12 pp 5757ndash5766 2004
[34] L Vitorino I M Chelo F Bacellar and L Ze-Ze ldquoRickettsiaephylogeny A multigenic approachrdquo Microbiology vol 153 no1 pp 160ndash168 2007
[35] J D Thompson T J Gibson F Plewniak F Jeanmougin andD G Higgins ldquoThe CLUSTAL X windows interface flexiblestrategies for multiple sequence alignment aided by qualityanalysis toolsrdquoNucleic Acids Research vol 25 no 24 pp 4876ndash4882 1997
[36] K Tamura G Stecher D Peterson A Filipski and S KumarldquoMEGA6 Molecular Evolutionary Genetics Analysis version60rdquoMolecular Biology and Evolution vol 30 no 12 pp 2725ndash2729 2013
[37] T H Ogden andM S Rosenberg ldquoHow should gaps be treatedin parsimony A comparison of approaches using simulationrdquoMolecular Phylogenetics and Evolution vol 42 no 3 pp 817ndash826 2007
[38] J P Huelsenbeck and F Ronquist ldquoMrBayes Bayesian inferenceof phylogenetic treesrdquo Bioinformatics vol 17 no 8 pp 754-7552001
[39] R W Emmons D V Dondero B C Nelson and R S LaneldquoEcology of tick-borne agents in Californiardquo The AmericanJournal of Tropical Medicine and Hygiene vol 30 no 1 pp 239ndash252 1981
[40] R C Pacheco M C Horta J Moraes-Filho A C Ataliba APinter and M B Labruna ldquoRickettsial infection in capybaras(Hyrochoerus hydrochaeris) from Sao Paulo Brazil Serologicalevidence for infection by Rickettsia bellii and Rickettsia parkerirdquoBiomedica vol 27 no 3 pp 364ndash371 2008
[41] M G Coelho V do Nascimento Ramos J E Limongi etal ldquoSerologic evidence of the exposure of small mammals tospotted-fever Rickettsia and Rickettsia bellii in Minas GeraisBrazilrdquoThe Journal of Infection in Developing Countries vol 10no 3 pp 275ndash282 2016
[42] RHu K EHyland andOliver ldquoA review on the use of ixodiph-agus wasps (Hymenoptera Encyrtidae) as natural enemies forthe control of ticks (Acari Ixodidae)rdquo Systematic and AppliedAcarology vol 3 pp 19ndash28 1998
[43] A J O Lopes J R S Nascimento-Junior C G Silva A PPrado M B Labruna and L M Costa-Junior ldquoParasitismby ixodiphagus wasps (Hymenoptera Encyrtidae) in Rhipi-cephalus sanguineus and Amblyomma ticks (Acari Ixodidae) inthree regions of Brazilrdquo Journal of Economic Entomology vol105 no 6 pp 1979ndash1981 2012
8 BioMed Research International
[44] M Bohacsova O Mediannikov M Kazimirova D Raoult andZ Sekeyova ldquoArsenophonus nasoniae and rickettsiae infectionof Ixodes ricinus due to parasitic wasp Ixodiphagus hookerirdquoPLoS ONE vol 11 no 2 Article ID e0149950 2016
[45] HOgata B La Scola S Audic et al ldquoGenome sequence ofRick-ettsia bellii illuminates the role of amoebae in gene exchangesbetween intracellular pathogensrdquo PLoS Genetics vol 2 no 5pp 733ndash744 2006
[46] S J Perlman M S Hunter and E Zchori-Fein ldquoThe emergingdiversity of Rickettsiardquo Proceedings of the Royal Society B Bio-logical Science vol 273 no 1598 pp 2097ndash2106 2006
[47] H Klompen and D Grimaldi ldquoFirst mesozoic record of aparasitiform mite A larval argasid tick in Cretaceous Amber(Acari Ixodida Argasidae)rdquoAnnals of the Entomological Societyof America vol 94 no 1 pp 10ndash15 2001
[48] D A Grimaldi M S Engel and P C Nascimbene ldquoFossilifer-ous cretaceous amber fromMyanmar (Burma) Its rediscoverybiotic diversity and paleontological significancerdquo AmericanMuseum Novitates vol 3361 pp 1ndash71 2002
[49] S Nava A A Guglielmone and A J Mangold ldquoAn overview ofsystematics and evolution of ticksrdquo Frontiers in Bioscience vol14 no 8 pp 2857ndash2877 2009
[50] A A Guglielmone D A Apanaskevich A Estrada-Pena R GRobbins T N Petney and I G Horak The Hard Ticks of theWorld (Acari Ixodida Ixodidae) vol 1 Springer Netherlandspress Dordrecht 1st edition 2014
Hindawiwwwhindawicom
International Journal of
Volume 2018
Zoology
Hindawiwwwhindawicom Volume 2018
Anatomy Research International
PeptidesInternational Journal of
Hindawiwwwhindawicom Volume 2018
Hindawiwwwhindawicom Volume 2018
Journal of Parasitology Research
GenomicsInternational Journal of
Hindawiwwwhindawicom Volume 2018
Hindawi Publishing Corporation httpwwwhindawicom Volume 2013Hindawiwwwhindawicom
The Scientific World Journal
Volume 2018
Hindawiwwwhindawicom Volume 2018
BioinformaticsAdvances in
Marine BiologyJournal of
Hindawiwwwhindawicom Volume 2018
Hindawiwwwhindawicom Volume 2018
Neuroscience Journal
Hindawiwwwhindawicom Volume 2018
BioMed Research International
Cell BiologyInternational Journal of
Hindawiwwwhindawicom Volume 2018
Hindawiwwwhindawicom Volume 2018
Biochemistry Research International
ArchaeaHindawiwwwhindawicom Volume 2018
Hindawiwwwhindawicom Volume 2018
Genetics Research International
Hindawiwwwhindawicom Volume 2018
Advances in
Virolog y Stem Cells International
Hindawiwwwhindawicom Volume 2018
Hindawiwwwhindawicom Volume 2018
Enzyme Research
Hindawiwwwhindawicom Volume 2018
International Journal of
MicrobiologyHindawiwwwhindawicom
Nucleic AcidsJournal of
Volume 2018
Submit your manuscripts atwwwhindawicom
2 BioMed Research International
Amblyomma ovale Amblyomma oblongoguttatum Ambly-omma parvum Amblyomma pseudoconcolor Amblyommarotundatum Amblyomma sabanerae Amblyomma scalptura-tum Amblyomma tigrinum andAmblyomma varium [6 7 1214ndash16]
The wide host range and extraordinarily broad distribu-tion of this Rickettsia species are intriguing and although Rbellii is generally considered as nonpathogenic for animalsand humans [2 17] it could play an important role in theecology and epidemiology of other pathogenic tick-borneSFG rickettsiae in the Americas [18] Despite the diverserepresentation of R bellii across more than 25 species of hardand soft ticks in the American continent the phylogeograph-ical relationships of this basal group-Rickettsia species hasnot been examined the work described here explores theserelationships based on a hypothesis that R bellii could haveevolved firstly in one continent or a particular tick group andthen radiated to other continents or other tick groups
2 Materials and Methods
21 Rickettsial Isolates Rickettsia bellii was isolated fromvarious species of hard ticks collected in South America andNorth America as described in the original publication ofeach of the isolates listed in Table 1 A total of 30 isolates of Rbellii from 13 species of hard ticks from 3 countries were usedfor the analysis (Figure 1) These included 1 from Argentina14 from Brazil and 15 from USA (Table 1) The Brazilian andArgentinean isolates are available at the Rickettsial Collectionof the Laboratory of Parasitic Diseases of Faculdade deMedicina Veterinaria e Zootecnia (FMVZ) of the Universityof Sao Paulo (USP) and the US isolates are available atthe Rickettsial Zoonoses Branch at the Centers for DiseaseControl and Prevention (CDC) Atlanta Georgia USA
22 DNAExtraction and PCR DNA from theR bellii isolatesfrom South America was extracted using the DNeasy Bloodand Tissue Kit (Qiagen Valencia CA) and the QIAampDNAMini Kit (Qiagen) was used for the 15 isolates from NorthAmerica all in accordance with the manufacturerrsquos recom-mendations Amplification of fragments of five rickettsialgenes and fifteen intergenic regions was attempted with theprimer pairs listed in Table 2 Each PCR reaction consistedof 2120583l template DNA 20 picomoles of each primer and10 120583l Taq PCRMaster Mix (Qiagen) while cycling conditionsincluded a 1-minute incubation at 95∘C followed by 35 cyclesof a 30-second denaturation at 95∘C a 30-second annealingincubation (Table 2) and a 1-minute extension at 72∘C Thiswas followed by a final 10-minute extension at 72∘C GradientPCR was used to optimize the annealing temperatures in Rbellii for each primer pair
23 DNA Purification and Sequencing DNA fragments am-plified by PCR were visualized using a UV lamp in a 15agarose gel containing 01120583gml ethidium bromide PCRproducts of the appropriate size were cut from the gel andthen purified using the Wizard SV gel and PCR clean-upsystem (Promega Madison WI) Sequencing reactions wereprepared using one microliter of purified PCR product and
the BigDye Terminator v31 Cycle Sequencing Kit (AppliedBiosystems Foster City CA) according to the manufacturerrsquosinstructions and then sequenced using an ABI 3100 geneticanalyzer (Applied Biosystems) Each PCR amplicon wassequenced at least once in both directions
24 Sequence Alignment and Phylogenetic Inferences Theresulting sequences were assembled using Geneious 914software (Biomatters Ltd Auckland New Zealand) andaligned using ClustalW [35] and MEGA 606 software [36]The resulting alignment was examined by eye to ensureproper alignment of the sequences and the simple indel-coding method [37] was used to remove insertionsdeletionsMrBayes 326 [38] was used in Geneious to perform aBayesian phylogenetic analysis The Jukes-Cantor model wasused in an analysis consisting of 10000000 generations(1000000 burn-ins) The analysis included 3 heated chainsand an effective sample size (ESS) of 901 was achieved for allparameters
25 AccessionNumbers TheGenBank accession numbers forthe DNA sequences generated in this study for the 30 Rbellii isolates shown in Table 1 are the following gltA gene(MF154866ndashMF154895) atpA gene (MF154926ndashMF154955)coxA gene (MF154896ndashMF154925) rpmE-tRNAfmet inter-genic region (MF154956ndashMF154985) and RC1027-xthA2intergenic region (MF154986ndashMF155015)
3 Results
Among the 20 primer pairs used for amplification of rick-ettsial DNA fragments only the primer pairs 15 16 1719 and 20 (Table 2) were successful in amplifying DNAfrom the R bellii isolates These primers corresponded to 3protein coding genes (gltA atpA and coxA) and 2 intergenicregions (rpmE-tRNAfmet and RC1027-xthA2)The nucleotidesequences from the five loci for each of the 30 isolates wereconcatenated and used to perform a Bayesian analysis oftheir phylogeny There was a separation of the isolates intoat least three clades one representing all South Americanisolates and the United States isolate CA-459 cultivated froma H leporispalustris tick collected in northern California[39] one comprising 6 isolates from D variabilis collectedin northern California [29] and a third clade comprising 5isolates from D variabilis collected in Ohio [30] (Figure 1)North American isolates from D parumapertus and the 369-C strain from D variabilis did not group within any of theabove clades Within the South American clade there weretwo smaller well-supported clades (each with a posteriorprobability of 10) one consisting of three isolates associatedwith I loricatus ticks and one consisting of two A ovale-associated isolates
Overall there were very few polymorphisms (lt05)between the 30 R bellii isolates In the 2269-nucleotidealignment identity values between the isolates varied from9956 to 100 (Table S1) DNA sequences of the isolatescomposing the main South American clade were 9969ndash100identical and at the same time 9956ndash9974 identical to theclade containing D variabilis isolates from California and
BioMed Research International 3
Minas Gerais 2 9
Rio Grande doSul 10 11
Mato Grosso do Sul 13
Ohio 23 25ndash27 29
California 17ndash22 30
Arkansas 16Utah 28
Texas 24
United States
Brazil
Argentina
IL-Mogi Ixodes loricatus (8)IL-RS1 Ixodes loricatus (10) RB-CL Ixodes loricatus (11)
AO Amblyomma ovale (6)P-10 Amblyomma ovale (14)
Mogi Amblyomma aureolatum (1) Guarda-Mor Amblyomma dubitatum (2) Ad-CORD-SP Amblyomma dubitatum (3) Ad-25-INT Amblyomma dubitatum (4) PNSM Amblyomma incisum (5)
HJ4 Haemaphysalis juxtakochi (7)
Ap GSV 136 Amblyomma parvum (9)HJ1 Haemaphysalis juxtakochi (12)
Rb MS PSEUDO Amblyomma pseudoconcolor (13) An4 Amblyomma neumanni (15)
CA-459 Haemaphysalis leporispalustris (30)
CA13-1 Dermacentor variabilis (17)CA13-9 Dermacentor variabilis (18)CA13-17 Dermacentor variabilis (19)Putah Creek Dermacentor variabilis (20)Yolo Dermacentor variabilis (21)Stevenson Bridge Dermacentor variabilis (22)
OSU 83-1223 Dermacentor variabilis (23) OSU 83-117 Dermacentor variabilis (25)OSU 85-1299 Dermacentor variabilis (26)OSU 83-452 Dermacentor variabilis (27) OSU 85-389 Dermacentor variabilis (29)
TX15-1 Dermacentor parumapertus (24)369C Dermacentor variabilis (16)
Skull Valley Dermacentor parumapertus (28)
1 3ndash8 12 14 So PauloCＩrdoba 15
20E minus 4
059
074
1
1
1
Figure 1 Molecular phylogenetic analysis of 30 isolates of Rickettsia bellii from North and South America
9974ndash9991 identical to the clade containing D variabilisisolates from Ohio Within-clade identities were 9974ndash100for the D variabilis isolates from Ohio and 100 for the Dvariabilis isolates from California the Ohio clade was 9974identical to the California clade Finally sequences of thethree North American isolates (two from D parumapertusand one from D variabilis) that did not group within anymain clade were 9987ndash9996 identical to each other and9960ndash9991 identical to the remaining isolates
4 Discussion
Here we performed for the first time a phylogenetic anal-ysis of multiple isolates of R bellii Because these isolatesrepresented a number of different locations in North andSouth America our intention was to infer phylogeographicalrelationships Indeed there was a separation of almost allisolates between the two continents suggesting that they haveradiated within their respective continents Although theposterior probability of the separation of the South Americanclade from the North American isolates was not strong (PP =059) this topology was also achieved using both the GTRand HKY85 evolutionary models (supplemental material)giving additional evidence for this separation At the sametime it is noteworthy that while the North American cladesrepresented isolates exclusively from Dermacentor ticks theSouth American clade was composed of isolates from thegenera Amblyomma Haemaphysalis and Ixodes Under suchcircumstances the phylogenetic positions of the 30 isolatesof the present study could be a result of not only theirgeographical origin but also the tick hosts in which theywere isolated from This later assumption is corroborated by
the position of the isolate from H leporispalustris whichdespite being from North America grouped within theSouth American clade where at least two other isolates fromHaemaphysalis ticks were present
To our knowledge the number and diversity of tickspecies infected with R bellii are the largest and broad-est described among species in the genus Rickettsia Themagnitude of different host species indicates horizontaltransmission among tick populations especially in SouthAmerica where isolates fromdifferent tick species and generagrouped together in the same clade It is generally acceptedthat R bellii is not pathogenic for vertebrates in fact Rbellii has never been isolated from a vertebrate host [2]On the other hand there has been serological evidence ofanimal natural infection or exposure by R bellii [40 41]While R bellii horizontal transmission among ticks via ver-tebrate host cannot be discarded another likely mechanismcould be via tick parasitoids since the parasitism of thehymenopteran Ixodiphagus spp is relatively common amongdifferent tick genera in the Americas [42 43] In fact a recentstudy provided molecular detection of tick-borne rickettsiaein Ixodiphagus wasps that had emerged from ticks [44]highlighting the possibility that rickettsial organisms couldbe shared by ticks and their parasitoids Regardless of themechanisms of horizontal transmission the tendency of eachR bellii genotype to be associated with a different tick species(Figure 1) suggests that horizontal transmission was moreefficient at earlier times thereaftermost of theR bellii isolatesare likely to have coevolved specifically with their specific tickspecies host possibly towards a symbiotic association In factanalysis of the genome of the type strain of R bellii has shownfeatures compatible with various symbiotic bacteria such as
4 BioMed Research International
Table 1 Rickettsia bellii isolates from South and North America used in the present study
Numberlowast Isolate name Tick host Geographic origin Reference(1) Mogi Amblyomma aureolatum Mogi das Cruzes-SP Brazil [19](2) Ad-MG Amblyomma dubitatum Guarda-Mor-MG Brazil [16](3) Ad-CORD-SP A dubitatum Cordeiropolis -SP Brazil [20](4) Ad-25-INT A dubitatum Ribeirao Grande-SP Brazil [20](5) PNSM Amblyomma incisum Cubatao-SP Brazil [21](6) AO Amblyomma ovale Ribeirao Grande-SP Brazil [22](7) HJ4 Haemaphysalis juxtakochi Ribeirao Grande-SP Brazil [23](8) IL-Mogi Ixodes loricatus Mogi das Cruzes-SP Brazil [24](9) Ap GSV 136 Amblyomma parvum Chapada Gaucha-MG Brazil [25](10) IL-RS1 I loricatus Derrubadas-RS Brazil [26](11) RB-CL I loricatus Cerro Largo-RS Brazil [13](12) HJ1 H juxtakochi Sao Paulo-SP Brazil [23](13) Ap-MS Amblyomma pseudoconcolor Corumba-MS Brazil [16](14) P-10 A ovale Peruıbe-SP Brazil [27](15) An4 Amblyomma neumanni Dean Funes-Cordoba Argentina [28](16) 369-C Dermacentor variabilis Washington Co Arkansas USA [5](17) CA13-1 Dermacentor variabilis Yolo Co California USA [29](18) CA13-9 D variabilis Yolo Co California USA [29](19) CA13-17 D variabilis Yolo Co California USA [29](20) Putah Creek D variabilis Solano Co California USA [29](21) Yolo D variabilis Yolo Co California USA [29](22) Stevenson Bridge D variabilis Yolo Co California USA [29](23) OSU 83-1223 D variabilis Knox County Ohio USA [30](24) TX15-1 Dermacentor parumapertus Brewster County Texas USA [31](25) OSU 83-117 D variabilis Licking County Ohio USA [30](26) OSU 85-1299 D variabilis Morrow County Ohio USA [30](27) OSU 83-452 D variabilis Coshocton County Ohio USA [30](28) Skull Valley D parumapertus Tooele County Utah USA [31](29) OSU 85-389 D variabilis Ohio USA [30](30) CA-459 Haemaphysalis leporispalustris Mendocino Co California USA [32]lowastThese numbers are represented in Figure 1 as the geographical location of each isolate
a large genome size with high coding capacity in contrastto the reduced genome size with low coding capacity of thepathogenic rickettsiae [4 45]
It has been proposed that the R bellii group divergedprior to the division between the SFG and the TG forming abasal group that also includes various herbivorous arthropodsymbionts [1 46] While the genus Rickettsia was consideredto be approximately 150 million years old the divergenceof the R bellii group was estimated to have occurred muchmore recently around 50 million years ago [1] Analysesof the oldest tick fossils from the Cretaceous indicate thatextant tick genera (Amblyomma Ornithodoros)were soundlyestablished around 100 million years ago [47ndash49] Fromthese data it can be inferred that R bellii likely radiatedwith its principal hosts approximately 50 million years agoWhile ticks and rickettsiae are distributed in all continentsit is noteworthy that under natural conditions and withouthuman interference tick species from the NewWorld do notoccur in the Old World and vice versa the only exceptionsare a few tick species that are associated with transcontinental
marine birds [50] Indeed this scenario has accounted forthe presence of R bellii-infected ticks restricted to the NewWorld regardless of the ability of this bacterium to infecta vast array of tick genera and species On the other handwhether R bellii originated with ticks in North or SouthAmerica remains obscure since our phylogenetic analysesdid not show any evidence for greater genetic divergenceof R bellii in any of the two continents Further analysesencompassing more R bellii isolates from different tickgenera and species encompassing more molecular markersshould provide cues for the origin and radiation of R bellii inthe NewWorld
5 Conclusion
Phylogeographical analysis of 30 strains (15 from NorthAmerica and 15 from South America) isolated from 13species of 4 genera shows a clear differentiation betweenmost of the North and South American isolates indicatinggeographic isolation between isolates of these two continents
BioMed Research International 5
Table2Prim
erpairs
used
fora
mplificatio
nof
rickettsialgenes
orintergenicregion
sinthep
resent
study
Prim
erpair
Target
Forw
ardprim
er(51015840to
31015840)
Reversep
rimer
(51015840to
31015840)
Ann
ealin
gtemperature
(∘ C)
Amplicon
size(nt)
Reference
(1)
mppA-purC
GCA
ATTA
TCGGTC
CGAAT
GTT
TCAT
TTAT
TTGTC
TCAAAAT
TCA
45to
551
160
[33]
(2)
nusG
-rplK
CAGTT
GCA
ATAT
TGGTA
AAG
CACA
GCA
GCT
GGAAT
TATC
AAG
TT45
to551
270
[33]
(3)
murG-RC0
563
GAAG
AAAAG
AAG
GGCA
TAAG
CTA
CAAG
CTGAAAG
TAAAAAC
ATTC
C40
to521
293
[33]
(4)
ntrY-rpsU
AGCT
GCT
GTT
GCT
AAAG
TAAAAA
CAAG
AAG
CAGCA
AGAAG
ACAG
A52
to641
363
[33]
(5)
dksA-xerC
TCCC
ATAG
GTA
ATTT
AGGTG
TTTC
TACT
ACCG
CATA
TCCA
ATTA
AAAA
45to
551
416
[33]
(6)
spo0J-a
bcT1
AAAG
ATTT
GGAAG
AAT
TAGAC
TTGAT
TTTG
CTTA
AAC
CAAC
CATT
TCA
45to
551
259
[33]
(7)
fabZ
-lpxD
TGTT
AGGAT
CGAT
TTTA
AGTA
CTCT
ATCT
TGGAT
TGGCA
TAGAC
AAT
CTAT
TA45
to551
195
[33]
(8)
RC1137-tlc-5
CGGGAT
AAC
GCC
GAG
TAAT
AAT
GCC
GCT
CTGAAT
TTGTT
T45
to551
264
[33]
(9)
RC0230-RC0
231
TGCA
CCCG
CCTA
AAAC
TAAC
ATGGTC
GGCC
GTA
GAAAAA
45to
551
232
[33]
(10)
groE
S-RC
0970
CTTG
CATC
GGCT
TTTC
TTTT
AGCT
TTGAG
CTGAT
GGGCT
A45
to551
215
[33]
(11)
rrf-p
yrH
GAG
CTTT
CTCC
ATCT
TTTC
TTG
AAAG
GGGAAT
ATAC
GAC
AAT
TGAG
45to
551
238
[33]
(12)
tRNA
Gly-tR
NA
Tyr
AGCT
TGGAAG
GCT
GGAAC
TCAT
CCTT
CTCC
CTCC
ACCA
CT45
to551
148
[33]
(13)
pal-R
C1201
TGCA
AGCA
CACA
TAAT
GCA
ATC
AAAAT
CGAT
TCCT
CTTT
TCC
45to
551
216
[33]
(14)
atpA
ATCA
AGCG
TTGCA
CAGAT
AGGGAAG
TGCC
GTA
AGTG
AAC
C58
Unk
nownlowast
[1]
(15)
rpmE-tRNA
fMet
TTCC
GGAAAT
GTA
GTA
AAT
CAAT
CTC
AGGTT
ATGAG
CCTG
ACGA
54144
[33]
(16)
RC1027-xthA2
GGTA
TGTA
AAT
GAG
CCTT
ATCA
ATAC
TTC
AGTA
GTA
TAAG
TAGCT
CCTG
CTGTC
54351
[33]
(17)
gltA
GTC
TACT
GCT
TCGTG
TAGAT
CAAC
GGCT
GAC
CTAT
AGAAT
ATTT
ATAAG
AC54
408
[25]
(18)
coxA
ACAG
CCGTT
GAT
ATGGCT
ACA
TATT
CCAAC
CGGCA
AAAG
58Unk
nownlowast
[1]
(19)
coxA
GGTG
CTCC
TGAT
ATGGCA
TTCA
TATT
CCAG
CCGGCA
AAAG
58Unk
nownlowast
[1]
(20)
atpA
ATCA
AGCG
TTGCA
CAGAT
AGCG
ACTT
ACCG
AAAT
ACCG
AC56
449
[134]
lowastDatan
otgivenin
theo
riginalpu
blication1teste
din
gradient
PCR
6 BioMed Research International
Additionally this analysis separated isolates of R bellii by thespecies of tick fromwhich these were isolated indicating thatthe isolates could have coevolved with their tick vectors overtime
Disclosure
The findings and conclusions are those of the authors anddo not necessarily reflect the views of the US Departmentof Health and Human Services or the official position of theCenters for Disease Control and Prevention
Conflicts of Interest
The authors declare that there are no conflicts of interestregarding the publication of this manuscript
Acknowledgments
This work received financial support from the Sao PauloResearch Foundation (FAPESP Grant 201510060-6)
Supplementary Materials
An identity matrix table of the 2269-nucleotide alignmentused for the phylogeny if R bellii is available in thesupplemental material for this article (Table S1) The finalalignment comprising 2269 nucleotides was concatenated inthe following order rpmE -tRNAfmet (414-nt) gltA (357-nt) coxA (898-nt) atpA (449-nt) and RC1027 -xthA2 (151-nt) and it is also available in the supplementary material(Rbellii final alignmentfas) Two additional phylogenetictrees are also available in the supplementary material (RbelliiGTR treepdf and Rbellii HKY treepdf) Table S1 identitymatrix of a 2269-nucleotide alignment of the 30 Rickettsiabellii isolates from Brazil (BRA) Argentina (ARG) and theUnited States (USA) used in the present study SupplementaryFigure 1 molecular phylogenetic analysis of 30 isolates ofRickettsia bellii from North and South America A total of2269 aligned nucleotide sites of 3 protein coding genes (gltAatpA and coxA) and 2 intergenic regions ( rpmE-tRNAfmet and RC1027- xthA2) were concatenated and subjectedto Bayesian analysis A total of 10000000 generations wererun using the GTRmodel with a sample frequency of 10000The analysis was run with 3 heated chains and the first1000000 generations were discarded as burn-in Numbers atnodes are support values derived from posterior probabilityThe scale bar is in units of expected substitutions per siteSupplementary Figure 2 molecular phylogenetic analysis of30 isolates of Rickettsia bellii fromNorth and South AmericaA total of 2269 aligned nucleotide sites of 3 protein codinggenes (gltA atpA and coxA) and 2 intergenic regions(rpmE-tRNA fmet and RC1027- xthA2) were concatenatedand subjected to Bayesian analysis A total of 10000000generations were run using the HKY85 model with a samplefrequency of 10000 The analysis was run with 3 heatedchains and the first 1000000 generations were discarded asburn-in Numbers at nodes are support values derived from
posterior probability The scale bar is in units of expectedsubstitutions per site (Supplementary Materials)
References
[1] L A Weinert J H Werren A Aebi G N Stone and F MJiggins ldquoEvolution and diversity of Rickettsia bacteriardquo BMCBiology vol 7 article no 6 2009
[2] P Parola C D Paddock C Socolovschi et al ldquoUpdate on tick-borne rickettsioses around the world a geographic approachrdquoClinical Microbiology Reviews vol 26 no 4 pp 657ndash702 2013
[3] D R Stothard J B Clark and P A Fuerst ldquoAncestral diver-gence of Rickettsia bellii from the spotted fever and typhusgroups of Rickettsia and antiquity of the genus Rickettsiardquo Inter-national Journal of Systematic Bacteriology vol 44 no 4 pp798ndash804 1994
[4] V Merhej and D Raoult ldquoRickettsial evolution in the light ofcomparative genomicsrdquo Biological Reviews vol 86 no 2 pp379ndash405 2011
[5] R N Philip E A Casper and R L Anacker ldquoRickettsia belliisp nov A tick-borne Rickettsia widely distributed in theUnited States that is distinct from the spotted fever and typhusbiogroupsrdquo International Journal of Systematic Bacteriology vol33 no 1 pp 94ndash106 1983
[6] M B Labruna S Mattar V and S Nava ldquoRickettsioses inLatin America Caribbean Spain and Portugalrdquo Revista MVZCordoba vol 16 no 2 pp 2435ndash2457 2011
[7] M Ogrzewalska I Literak J M Cardenas-CallirgosM Capekand M B Labruna ldquoRickettsia bellii in ticks Amblyommavarium Koch 1844 from birds in Perurdquo Ticks and Tick-borneDiseases vol 3 no 4 pp 254ndash256 2012
[8] M Ogrzewalska D G Saraiva J Moraes-Filho et al ldquoEpidemi-ology of Brazilian spotted fever in the Atlantic Forest state ofSao Paulo Brazilrdquo Parasitology vol 139 no 10 pp 1283ndash13002012
[9] J Miranda and S Mattar ldquoMolecular detection of Rickettsiabellii and Rickettsia sp strain Colombianensi in ticks fromCordoba Colombiardquo Ticks and Tick-borne Diseases vol 5 no2 pp 208ndash212 2014
[10] M Andoh A Sakata A Takano et al ldquoDetection of Rickettsiaand Ehrlichia spp In ticks associated with exotic reptiles andamphibians imported into Japanrdquo PLoS ONE vol 10 no 7Article ID e0133700 2015
[11] M Ogrzewalska I Literak M Capek et al ldquoBacteria of thegenus Rickettsia in ticks (Acari Ixodidae) collected from birdsin Costa Ricardquo Ticks and Tick-borne Diseases vol 6 no 4 pp478ndash482 2015
[12] A R M Barbieri L Romero and M B Labruna ldquoRickettsiabellii infecting Amblyomma sabanerae ticks in El SalvadorrdquoPathogens and Global Health vol 106 no 3 pp 188-189 2012
[13] F S Krawczak L C Binder C S Oliveira et al ldquoEcology of atick-borne spotted fever in southern Brazilrdquo Experimental andApplied Acarology vol 70 no 2 pp 219ndash229 2016
[14] D McIntosh R A Bezerra H R Luz et al ldquoDetectionof Rickettsia bellii and Rickettsia amblyommii in Amblyommalongirostre (Acari Ixodidae) fromBahia state Northeast BrazilrdquoBrazilian Journal of Microbiology vol 46 no 3 pp 879ndash8832015
[15] H S Soares A R M Barbieri T F Martins et al ldquoTicks andrickettsial infection in thewildlife of two regions of the BrazilianAmazonrdquo Experimental and Applied Acarology vol 65 no 1 pp125ndash140 2015
BioMed Research International 7
[16] F B Costa A R M Barbieri M P J Szabo V N Ramos UPiovezan and M B Labruna ldquoNew records of Rickettsia bellii-infected ticks in Brazilrdquo Brazilian Journal of Veterinary Researchand Animal Science vol 54 no 1 pp 92ndash95 2017
[17] M B Labruna T Whitworth M C Horta et al ldquoRickettsiaspecies infecting Amblyomma cooperi ticks from an area inthe state of Sao Paulo Brazil where Brazilian Spotted Fever isendemicrdquo Journal of Clinical Microbiology vol 42 no 1 pp 90ndash98 2004
[18] K R Macaluso D E Sonenshine S M Ceraul and A F AzadldquoRickettsial infection in Dermacentor variabilis (Acari Ixodi-dae) inhibits transovarial transmission of a second RickettsiardquoJournal of Medical Entomology vol 39 no 6 pp 809ndash813 2002
[19] A Pinter and M B Labruna ldquoIsolation of Rickettsia rickettsiiand Rickettsia bellii in cell culture from the tick Amblyommaaureolatum in Brazilrdquo Annals of the New York Academy ofSciences vol 1078 pp 523ndash529 2006
[20] R De Campos Pacheco M C Horta A Pinter et al ldquoSurvey ofRickettsia spp in the ticks Amblyomma cajennense and Ambly-omma dubitatum in the State of Sao Paulordquo Journal of theBrazilian Society of Tropical Medicine vol 42 no 3 pp 351ndash3532009
[21] G S Sabatini Pesquisa de Carrapatos e Riquetsias no ParqueEstadual da Serra do Mar Nucleo Itutinga ndash Pilloes Sao Paulo[Dissertation thesis] University of Sao Paulo Sao Paulo SP2010
[22] R Pacheco S Rosa L Richtzenhain M P J Szabo and M BLabruna ldquoIsolation of Rickettsia bellii from Amblyomma ovaleand Amblyomma incisum ticks from southern Brazilrdquo RevistaMVZ Cordoba vol 13 no 2 pp 1273ndash1279 2008
[23] M B Labruna R C Pacheco L J Richtzenhain and M P JSzabo ldquoIsolation of Rickettsia rhipicephali and Rickettsia belliifrom Haemaphysalis juxtakochi ticks in the state of Sao PauloBrazilrdquo Applied and Environmental Microbiology vol 73 no 3pp 869ndash873 2007
[24] M C Horta A Pinter T T S Schumaker and M B LabrunaldquoNatural infection transovarial transmission and transstadialsurvival of Rickettsia bellii in the tick Ixodes loricatus (AcariIxodidae) from Brazilrdquo Annals of the New York Academy ofSciences vol 1078 pp 285ndash290 2006
[25] A R M Barbieri Ecologia de carrapatos e rickettsias transmiti-das por carrapatos em uma reserva natural de Cerrado brasileiro[PhD thesis] University of Sao Paulo Sao Paulo SP 2016
[26] F S KrawczakPhD thesis University of Sao Paulo Sao Paulo SPPesquisa de infeccao por riquetsias do grupo da febre maculosaem caes pequenos mamıferos e carrapatos em area endemica enao endemicas nos biomas Pampa eMata Atlantica no estado doRio Grande do Sul [PhD thesis] University of Sao Paulo SaoPaulo SP 2016
[27] M P J Szabo F A Nieri-BastosM G Spolidorio T FMartinsA M Barbieri and M B Labruna ldquoIn vitro isolation fromAmblyomma ovale (Acari Ixodidae) and ecological aspects ofthe Atlantic rainforest Rickettsia the causative agent of a novelspotted fever rickettsiosis in Brazilrdquo Parasitology vol 140 no 6pp 719ndash728 2013
[28] M B Labruna R C Pacheco S Nava P E Brandao L JRichtzenhain and A A Guglielmone ldquoInfection by Rickettsiabellii and Candidatus ldquoRickettsia amblyommiirdquo in Amblyommaneumanni ticks from ArgentinardquoMicrobial Ecology vol 54 no1 pp 126ndash133 2007
[29] J A Hecht M E J Allerdice F S Krawczak M B Labruna CD Paddock and S E Karpathy ldquoDevelopment of a Rickettsia
bellii-specific TaqMan assay targeting the citrate synthase generdquoJournal of Medical Entomology vol 53 no 6 pp 1492ndash14952016
[30] K Poetter PhD thesis Molecular population and evolutionarygenetics of Rickettsia Ohio State University Columbus OHUSA 1989
[31] C D Paddock M E J Allerdice S E Karpathy et al ldquoUniquestrain of Rickettsia parkeri associated with the hard tickDerma-centor parumapertus Neumann in the western United StatesrdquoApplied and Environmental Microbiology vol 83 no 9 ArticleID e03463-16 2017
[32] R N Philip E A Casper and R S Lane ldquoSerotypes of tick-borne spotted fever group Rickettsiae from western CaliforniardquoTheAmerican Journal of Tropical Medicine and Hygiene vol 30no 3 pp 722ndash727 1981
[33] P-E Fournier Y Zhu H Ogata and D Raoult ldquoUse of highlyvariable intergenic spacer sequences for multispacer typing ofRickettsia conorii strainsrdquo Journal of Clinical Microbiology vol42 no 12 pp 5757ndash5766 2004
[34] L Vitorino I M Chelo F Bacellar and L Ze-Ze ldquoRickettsiaephylogeny A multigenic approachrdquo Microbiology vol 153 no1 pp 160ndash168 2007
[35] J D Thompson T J Gibson F Plewniak F Jeanmougin andD G Higgins ldquoThe CLUSTAL X windows interface flexiblestrategies for multiple sequence alignment aided by qualityanalysis toolsrdquoNucleic Acids Research vol 25 no 24 pp 4876ndash4882 1997
[36] K Tamura G Stecher D Peterson A Filipski and S KumarldquoMEGA6 Molecular Evolutionary Genetics Analysis version60rdquoMolecular Biology and Evolution vol 30 no 12 pp 2725ndash2729 2013
[37] T H Ogden andM S Rosenberg ldquoHow should gaps be treatedin parsimony A comparison of approaches using simulationrdquoMolecular Phylogenetics and Evolution vol 42 no 3 pp 817ndash826 2007
[38] J P Huelsenbeck and F Ronquist ldquoMrBayes Bayesian inferenceof phylogenetic treesrdquo Bioinformatics vol 17 no 8 pp 754-7552001
[39] R W Emmons D V Dondero B C Nelson and R S LaneldquoEcology of tick-borne agents in Californiardquo The AmericanJournal of Tropical Medicine and Hygiene vol 30 no 1 pp 239ndash252 1981
[40] R C Pacheco M C Horta J Moraes-Filho A C Ataliba APinter and M B Labruna ldquoRickettsial infection in capybaras(Hyrochoerus hydrochaeris) from Sao Paulo Brazil Serologicalevidence for infection by Rickettsia bellii and Rickettsia parkerirdquoBiomedica vol 27 no 3 pp 364ndash371 2008
[41] M G Coelho V do Nascimento Ramos J E Limongi etal ldquoSerologic evidence of the exposure of small mammals tospotted-fever Rickettsia and Rickettsia bellii in Minas GeraisBrazilrdquoThe Journal of Infection in Developing Countries vol 10no 3 pp 275ndash282 2016
[42] RHu K EHyland andOliver ldquoA review on the use of ixodiph-agus wasps (Hymenoptera Encyrtidae) as natural enemies forthe control of ticks (Acari Ixodidae)rdquo Systematic and AppliedAcarology vol 3 pp 19ndash28 1998
[43] A J O Lopes J R S Nascimento-Junior C G Silva A PPrado M B Labruna and L M Costa-Junior ldquoParasitismby ixodiphagus wasps (Hymenoptera Encyrtidae) in Rhipi-cephalus sanguineus and Amblyomma ticks (Acari Ixodidae) inthree regions of Brazilrdquo Journal of Economic Entomology vol105 no 6 pp 1979ndash1981 2012
8 BioMed Research International
[44] M Bohacsova O Mediannikov M Kazimirova D Raoult andZ Sekeyova ldquoArsenophonus nasoniae and rickettsiae infectionof Ixodes ricinus due to parasitic wasp Ixodiphagus hookerirdquoPLoS ONE vol 11 no 2 Article ID e0149950 2016
[45] HOgata B La Scola S Audic et al ldquoGenome sequence ofRick-ettsia bellii illuminates the role of amoebae in gene exchangesbetween intracellular pathogensrdquo PLoS Genetics vol 2 no 5pp 733ndash744 2006
[46] S J Perlman M S Hunter and E Zchori-Fein ldquoThe emergingdiversity of Rickettsiardquo Proceedings of the Royal Society B Bio-logical Science vol 273 no 1598 pp 2097ndash2106 2006
[47] H Klompen and D Grimaldi ldquoFirst mesozoic record of aparasitiform mite A larval argasid tick in Cretaceous Amber(Acari Ixodida Argasidae)rdquoAnnals of the Entomological Societyof America vol 94 no 1 pp 10ndash15 2001
[48] D A Grimaldi M S Engel and P C Nascimbene ldquoFossilifer-ous cretaceous amber fromMyanmar (Burma) Its rediscoverybiotic diversity and paleontological significancerdquo AmericanMuseum Novitates vol 3361 pp 1ndash71 2002
[49] S Nava A A Guglielmone and A J Mangold ldquoAn overview ofsystematics and evolution of ticksrdquo Frontiers in Bioscience vol14 no 8 pp 2857ndash2877 2009
[50] A A Guglielmone D A Apanaskevich A Estrada-Pena R GRobbins T N Petney and I G Horak The Hard Ticks of theWorld (Acari Ixodida Ixodidae) vol 1 Springer Netherlandspress Dordrecht 1st edition 2014
Hindawiwwwhindawicom
International Journal of
Volume 2018
Zoology
Hindawiwwwhindawicom Volume 2018
Anatomy Research International
PeptidesInternational Journal of
Hindawiwwwhindawicom Volume 2018
Hindawiwwwhindawicom Volume 2018
Journal of Parasitology Research
GenomicsInternational Journal of
Hindawiwwwhindawicom Volume 2018
Hindawi Publishing Corporation httpwwwhindawicom Volume 2013Hindawiwwwhindawicom
The Scientific World Journal
Volume 2018
Hindawiwwwhindawicom Volume 2018
BioinformaticsAdvances in
Marine BiologyJournal of
Hindawiwwwhindawicom Volume 2018
Hindawiwwwhindawicom Volume 2018
Neuroscience Journal
Hindawiwwwhindawicom Volume 2018
BioMed Research International
Cell BiologyInternational Journal of
Hindawiwwwhindawicom Volume 2018
Hindawiwwwhindawicom Volume 2018
Biochemistry Research International
ArchaeaHindawiwwwhindawicom Volume 2018
Hindawiwwwhindawicom Volume 2018
Genetics Research International
Hindawiwwwhindawicom Volume 2018
Advances in
Virolog y Stem Cells International
Hindawiwwwhindawicom Volume 2018
Hindawiwwwhindawicom Volume 2018
Enzyme Research
Hindawiwwwhindawicom Volume 2018
International Journal of
MicrobiologyHindawiwwwhindawicom
Nucleic AcidsJournal of
Volume 2018
Submit your manuscripts atwwwhindawicom
BioMed Research International 3
Minas Gerais 2 9
Rio Grande doSul 10 11
Mato Grosso do Sul 13
Ohio 23 25ndash27 29
California 17ndash22 30
Arkansas 16Utah 28
Texas 24
United States
Brazil
Argentina
IL-Mogi Ixodes loricatus (8)IL-RS1 Ixodes loricatus (10) RB-CL Ixodes loricatus (11)
AO Amblyomma ovale (6)P-10 Amblyomma ovale (14)
Mogi Amblyomma aureolatum (1) Guarda-Mor Amblyomma dubitatum (2) Ad-CORD-SP Amblyomma dubitatum (3) Ad-25-INT Amblyomma dubitatum (4) PNSM Amblyomma incisum (5)
HJ4 Haemaphysalis juxtakochi (7)
Ap GSV 136 Amblyomma parvum (9)HJ1 Haemaphysalis juxtakochi (12)
Rb MS PSEUDO Amblyomma pseudoconcolor (13) An4 Amblyomma neumanni (15)
CA-459 Haemaphysalis leporispalustris (30)
CA13-1 Dermacentor variabilis (17)CA13-9 Dermacentor variabilis (18)CA13-17 Dermacentor variabilis (19)Putah Creek Dermacentor variabilis (20)Yolo Dermacentor variabilis (21)Stevenson Bridge Dermacentor variabilis (22)
OSU 83-1223 Dermacentor variabilis (23) OSU 83-117 Dermacentor variabilis (25)OSU 85-1299 Dermacentor variabilis (26)OSU 83-452 Dermacentor variabilis (27) OSU 85-389 Dermacentor variabilis (29)
TX15-1 Dermacentor parumapertus (24)369C Dermacentor variabilis (16)
Skull Valley Dermacentor parumapertus (28)
1 3ndash8 12 14 So PauloCＩrdoba 15
20E minus 4
059
074
1
1
1
Figure 1 Molecular phylogenetic analysis of 30 isolates of Rickettsia bellii from North and South America
9974ndash9991 identical to the clade containing D variabilisisolates from Ohio Within-clade identities were 9974ndash100for the D variabilis isolates from Ohio and 100 for the Dvariabilis isolates from California the Ohio clade was 9974identical to the California clade Finally sequences of thethree North American isolates (two from D parumapertusand one from D variabilis) that did not group within anymain clade were 9987ndash9996 identical to each other and9960ndash9991 identical to the remaining isolates
4 Discussion
Here we performed for the first time a phylogenetic anal-ysis of multiple isolates of R bellii Because these isolatesrepresented a number of different locations in North andSouth America our intention was to infer phylogeographicalrelationships Indeed there was a separation of almost allisolates between the two continents suggesting that they haveradiated within their respective continents Although theposterior probability of the separation of the South Americanclade from the North American isolates was not strong (PP =059) this topology was also achieved using both the GTRand HKY85 evolutionary models (supplemental material)giving additional evidence for this separation At the sametime it is noteworthy that while the North American cladesrepresented isolates exclusively from Dermacentor ticks theSouth American clade was composed of isolates from thegenera Amblyomma Haemaphysalis and Ixodes Under suchcircumstances the phylogenetic positions of the 30 isolatesof the present study could be a result of not only theirgeographical origin but also the tick hosts in which theywere isolated from This later assumption is corroborated by
the position of the isolate from H leporispalustris whichdespite being from North America grouped within theSouth American clade where at least two other isolates fromHaemaphysalis ticks were present
To our knowledge the number and diversity of tickspecies infected with R bellii are the largest and broad-est described among species in the genus Rickettsia Themagnitude of different host species indicates horizontaltransmission among tick populations especially in SouthAmerica where isolates fromdifferent tick species and generagrouped together in the same clade It is generally acceptedthat R bellii is not pathogenic for vertebrates in fact Rbellii has never been isolated from a vertebrate host [2]On the other hand there has been serological evidence ofanimal natural infection or exposure by R bellii [40 41]While R bellii horizontal transmission among ticks via ver-tebrate host cannot be discarded another likely mechanismcould be via tick parasitoids since the parasitism of thehymenopteran Ixodiphagus spp is relatively common amongdifferent tick genera in the Americas [42 43] In fact a recentstudy provided molecular detection of tick-borne rickettsiaein Ixodiphagus wasps that had emerged from ticks [44]highlighting the possibility that rickettsial organisms couldbe shared by ticks and their parasitoids Regardless of themechanisms of horizontal transmission the tendency of eachR bellii genotype to be associated with a different tick species(Figure 1) suggests that horizontal transmission was moreefficient at earlier times thereaftermost of theR bellii isolatesare likely to have coevolved specifically with their specific tickspecies host possibly towards a symbiotic association In factanalysis of the genome of the type strain of R bellii has shownfeatures compatible with various symbiotic bacteria such as
4 BioMed Research International
Table 1 Rickettsia bellii isolates from South and North America used in the present study
Numberlowast Isolate name Tick host Geographic origin Reference(1) Mogi Amblyomma aureolatum Mogi das Cruzes-SP Brazil [19](2) Ad-MG Amblyomma dubitatum Guarda-Mor-MG Brazil [16](3) Ad-CORD-SP A dubitatum Cordeiropolis -SP Brazil [20](4) Ad-25-INT A dubitatum Ribeirao Grande-SP Brazil [20](5) PNSM Amblyomma incisum Cubatao-SP Brazil [21](6) AO Amblyomma ovale Ribeirao Grande-SP Brazil [22](7) HJ4 Haemaphysalis juxtakochi Ribeirao Grande-SP Brazil [23](8) IL-Mogi Ixodes loricatus Mogi das Cruzes-SP Brazil [24](9) Ap GSV 136 Amblyomma parvum Chapada Gaucha-MG Brazil [25](10) IL-RS1 I loricatus Derrubadas-RS Brazil [26](11) RB-CL I loricatus Cerro Largo-RS Brazil [13](12) HJ1 H juxtakochi Sao Paulo-SP Brazil [23](13) Ap-MS Amblyomma pseudoconcolor Corumba-MS Brazil [16](14) P-10 A ovale Peruıbe-SP Brazil [27](15) An4 Amblyomma neumanni Dean Funes-Cordoba Argentina [28](16) 369-C Dermacentor variabilis Washington Co Arkansas USA [5](17) CA13-1 Dermacentor variabilis Yolo Co California USA [29](18) CA13-9 D variabilis Yolo Co California USA [29](19) CA13-17 D variabilis Yolo Co California USA [29](20) Putah Creek D variabilis Solano Co California USA [29](21) Yolo D variabilis Yolo Co California USA [29](22) Stevenson Bridge D variabilis Yolo Co California USA [29](23) OSU 83-1223 D variabilis Knox County Ohio USA [30](24) TX15-1 Dermacentor parumapertus Brewster County Texas USA [31](25) OSU 83-117 D variabilis Licking County Ohio USA [30](26) OSU 85-1299 D variabilis Morrow County Ohio USA [30](27) OSU 83-452 D variabilis Coshocton County Ohio USA [30](28) Skull Valley D parumapertus Tooele County Utah USA [31](29) OSU 85-389 D variabilis Ohio USA [30](30) CA-459 Haemaphysalis leporispalustris Mendocino Co California USA [32]lowastThese numbers are represented in Figure 1 as the geographical location of each isolate
a large genome size with high coding capacity in contrastto the reduced genome size with low coding capacity of thepathogenic rickettsiae [4 45]
It has been proposed that the R bellii group divergedprior to the division between the SFG and the TG forming abasal group that also includes various herbivorous arthropodsymbionts [1 46] While the genus Rickettsia was consideredto be approximately 150 million years old the divergenceof the R bellii group was estimated to have occurred muchmore recently around 50 million years ago [1] Analysesof the oldest tick fossils from the Cretaceous indicate thatextant tick genera (Amblyomma Ornithodoros)were soundlyestablished around 100 million years ago [47ndash49] Fromthese data it can be inferred that R bellii likely radiatedwith its principal hosts approximately 50 million years agoWhile ticks and rickettsiae are distributed in all continentsit is noteworthy that under natural conditions and withouthuman interference tick species from the NewWorld do notoccur in the Old World and vice versa the only exceptionsare a few tick species that are associated with transcontinental
marine birds [50] Indeed this scenario has accounted forthe presence of R bellii-infected ticks restricted to the NewWorld regardless of the ability of this bacterium to infecta vast array of tick genera and species On the other handwhether R bellii originated with ticks in North or SouthAmerica remains obscure since our phylogenetic analysesdid not show any evidence for greater genetic divergenceof R bellii in any of the two continents Further analysesencompassing more R bellii isolates from different tickgenera and species encompassing more molecular markersshould provide cues for the origin and radiation of R bellii inthe NewWorld
5 Conclusion
Phylogeographical analysis of 30 strains (15 from NorthAmerica and 15 from South America) isolated from 13species of 4 genera shows a clear differentiation betweenmost of the North and South American isolates indicatinggeographic isolation between isolates of these two continents
BioMed Research International 5
Table2Prim
erpairs
used
fora
mplificatio
nof
rickettsialgenes
orintergenicregion
sinthep
resent
study
Prim
erpair
Target
Forw
ardprim
er(51015840to
31015840)
Reversep
rimer
(51015840to
31015840)
Ann
ealin
gtemperature
(∘ C)
Amplicon
size(nt)
Reference
(1)
mppA-purC
GCA
ATTA
TCGGTC
CGAAT
GTT
TCAT
TTAT
TTGTC
TCAAAAT
TCA
45to
551
160
[33]
(2)
nusG
-rplK
CAGTT
GCA
ATAT
TGGTA
AAG
CACA
GCA
GCT
GGAAT
TATC
AAG
TT45
to551
270
[33]
(3)
murG-RC0
563
GAAG
AAAAG
AAG
GGCA
TAAG
CTA
CAAG
CTGAAAG
TAAAAAC
ATTC
C40
to521
293
[33]
(4)
ntrY-rpsU
AGCT
GCT
GTT
GCT
AAAG
TAAAAA
CAAG
AAG
CAGCA
AGAAG
ACAG
A52
to641
363
[33]
(5)
dksA-xerC
TCCC
ATAG
GTA
ATTT
AGGTG
TTTC
TACT
ACCG
CATA
TCCA
ATTA
AAAA
45to
551
416
[33]
(6)
spo0J-a
bcT1
AAAG
ATTT
GGAAG
AAT
TAGAC
TTGAT
TTTG
CTTA
AAC
CAAC
CATT
TCA
45to
551
259
[33]
(7)
fabZ
-lpxD
TGTT
AGGAT
CGAT
TTTA
AGTA
CTCT
ATCT
TGGAT
TGGCA
TAGAC
AAT
CTAT
TA45
to551
195
[33]
(8)
RC1137-tlc-5
CGGGAT
AAC
GCC
GAG
TAAT
AAT
GCC
GCT
CTGAAT
TTGTT
T45
to551
264
[33]
(9)
RC0230-RC0
231
TGCA
CCCG
CCTA
AAAC
TAAC
ATGGTC
GGCC
GTA
GAAAAA
45to
551
232
[33]
(10)
groE
S-RC
0970
CTTG
CATC
GGCT
TTTC
TTTT
AGCT
TTGAG
CTGAT
GGGCT
A45
to551
215
[33]
(11)
rrf-p
yrH
GAG
CTTT
CTCC
ATCT
TTTC
TTG
AAAG
GGGAAT
ATAC
GAC
AAT
TGAG
45to
551
238
[33]
(12)
tRNA
Gly-tR
NA
Tyr
AGCT
TGGAAG
GCT
GGAAC
TCAT
CCTT
CTCC
CTCC
ACCA
CT45
to551
148
[33]
(13)
pal-R
C1201
TGCA
AGCA
CACA
TAAT
GCA
ATC
AAAAT
CGAT
TCCT
CTTT
TCC
45to
551
216
[33]
(14)
atpA
ATCA
AGCG
TTGCA
CAGAT
AGGGAAG
TGCC
GTA
AGTG
AAC
C58
Unk
nownlowast
[1]
(15)
rpmE-tRNA
fMet
TTCC
GGAAAT
GTA
GTA
AAT
CAAT
CTC
AGGTT
ATGAG
CCTG
ACGA
54144
[33]
(16)
RC1027-xthA2
GGTA
TGTA
AAT
GAG
CCTT
ATCA
ATAC
TTC
AGTA
GTA
TAAG
TAGCT
CCTG
CTGTC
54351
[33]
(17)
gltA
GTC
TACT
GCT
TCGTG
TAGAT
CAAC
GGCT
GAC
CTAT
AGAAT
ATTT
ATAAG
AC54
408
[25]
(18)
coxA
ACAG
CCGTT
GAT
ATGGCT
ACA
TATT
CCAAC
CGGCA
AAAG
58Unk
nownlowast
[1]
(19)
coxA
GGTG
CTCC
TGAT
ATGGCA
TTCA
TATT
CCAG
CCGGCA
AAAG
58Unk
nownlowast
[1]
(20)
atpA
ATCA
AGCG
TTGCA
CAGAT
AGCG
ACTT
ACCG
AAAT
ACCG
AC56
449
[134]
lowastDatan
otgivenin
theo
riginalpu
blication1teste
din
gradient
PCR
6 BioMed Research International
Additionally this analysis separated isolates of R bellii by thespecies of tick fromwhich these were isolated indicating thatthe isolates could have coevolved with their tick vectors overtime
Disclosure
The findings and conclusions are those of the authors anddo not necessarily reflect the views of the US Departmentof Health and Human Services or the official position of theCenters for Disease Control and Prevention
Conflicts of Interest
The authors declare that there are no conflicts of interestregarding the publication of this manuscript
Acknowledgments
This work received financial support from the Sao PauloResearch Foundation (FAPESP Grant 201510060-6)
Supplementary Materials
An identity matrix table of the 2269-nucleotide alignmentused for the phylogeny if R bellii is available in thesupplemental material for this article (Table S1) The finalalignment comprising 2269 nucleotides was concatenated inthe following order rpmE -tRNAfmet (414-nt) gltA (357-nt) coxA (898-nt) atpA (449-nt) and RC1027 -xthA2 (151-nt) and it is also available in the supplementary material(Rbellii final alignmentfas) Two additional phylogenetictrees are also available in the supplementary material (RbelliiGTR treepdf and Rbellii HKY treepdf) Table S1 identitymatrix of a 2269-nucleotide alignment of the 30 Rickettsiabellii isolates from Brazil (BRA) Argentina (ARG) and theUnited States (USA) used in the present study SupplementaryFigure 1 molecular phylogenetic analysis of 30 isolates ofRickettsia bellii from North and South America A total of2269 aligned nucleotide sites of 3 protein coding genes (gltAatpA and coxA) and 2 intergenic regions ( rpmE-tRNAfmet and RC1027- xthA2) were concatenated and subjectedto Bayesian analysis A total of 10000000 generations wererun using the GTRmodel with a sample frequency of 10000The analysis was run with 3 heated chains and the first1000000 generations were discarded as burn-in Numbers atnodes are support values derived from posterior probabilityThe scale bar is in units of expected substitutions per siteSupplementary Figure 2 molecular phylogenetic analysis of30 isolates of Rickettsia bellii fromNorth and South AmericaA total of 2269 aligned nucleotide sites of 3 protein codinggenes (gltA atpA and coxA) and 2 intergenic regions(rpmE-tRNA fmet and RC1027- xthA2) were concatenatedand subjected to Bayesian analysis A total of 10000000generations were run using the HKY85 model with a samplefrequency of 10000 The analysis was run with 3 heatedchains and the first 1000000 generations were discarded asburn-in Numbers at nodes are support values derived from
posterior probability The scale bar is in units of expectedsubstitutions per site (Supplementary Materials)
References
[1] L A Weinert J H Werren A Aebi G N Stone and F MJiggins ldquoEvolution and diversity of Rickettsia bacteriardquo BMCBiology vol 7 article no 6 2009
[2] P Parola C D Paddock C Socolovschi et al ldquoUpdate on tick-borne rickettsioses around the world a geographic approachrdquoClinical Microbiology Reviews vol 26 no 4 pp 657ndash702 2013
[3] D R Stothard J B Clark and P A Fuerst ldquoAncestral diver-gence of Rickettsia bellii from the spotted fever and typhusgroups of Rickettsia and antiquity of the genus Rickettsiardquo Inter-national Journal of Systematic Bacteriology vol 44 no 4 pp798ndash804 1994
[4] V Merhej and D Raoult ldquoRickettsial evolution in the light ofcomparative genomicsrdquo Biological Reviews vol 86 no 2 pp379ndash405 2011
[5] R N Philip E A Casper and R L Anacker ldquoRickettsia belliisp nov A tick-borne Rickettsia widely distributed in theUnited States that is distinct from the spotted fever and typhusbiogroupsrdquo International Journal of Systematic Bacteriology vol33 no 1 pp 94ndash106 1983
[6] M B Labruna S Mattar V and S Nava ldquoRickettsioses inLatin America Caribbean Spain and Portugalrdquo Revista MVZCordoba vol 16 no 2 pp 2435ndash2457 2011
[7] M Ogrzewalska I Literak J M Cardenas-CallirgosM Capekand M B Labruna ldquoRickettsia bellii in ticks Amblyommavarium Koch 1844 from birds in Perurdquo Ticks and Tick-borneDiseases vol 3 no 4 pp 254ndash256 2012
[8] M Ogrzewalska D G Saraiva J Moraes-Filho et al ldquoEpidemi-ology of Brazilian spotted fever in the Atlantic Forest state ofSao Paulo Brazilrdquo Parasitology vol 139 no 10 pp 1283ndash13002012
[9] J Miranda and S Mattar ldquoMolecular detection of Rickettsiabellii and Rickettsia sp strain Colombianensi in ticks fromCordoba Colombiardquo Ticks and Tick-borne Diseases vol 5 no2 pp 208ndash212 2014
[10] M Andoh A Sakata A Takano et al ldquoDetection of Rickettsiaand Ehrlichia spp In ticks associated with exotic reptiles andamphibians imported into Japanrdquo PLoS ONE vol 10 no 7Article ID e0133700 2015
[11] M Ogrzewalska I Literak M Capek et al ldquoBacteria of thegenus Rickettsia in ticks (Acari Ixodidae) collected from birdsin Costa Ricardquo Ticks and Tick-borne Diseases vol 6 no 4 pp478ndash482 2015
[12] A R M Barbieri L Romero and M B Labruna ldquoRickettsiabellii infecting Amblyomma sabanerae ticks in El SalvadorrdquoPathogens and Global Health vol 106 no 3 pp 188-189 2012
[13] F S Krawczak L C Binder C S Oliveira et al ldquoEcology of atick-borne spotted fever in southern Brazilrdquo Experimental andApplied Acarology vol 70 no 2 pp 219ndash229 2016
[14] D McIntosh R A Bezerra H R Luz et al ldquoDetectionof Rickettsia bellii and Rickettsia amblyommii in Amblyommalongirostre (Acari Ixodidae) fromBahia state Northeast BrazilrdquoBrazilian Journal of Microbiology vol 46 no 3 pp 879ndash8832015
[15] H S Soares A R M Barbieri T F Martins et al ldquoTicks andrickettsial infection in thewildlife of two regions of the BrazilianAmazonrdquo Experimental and Applied Acarology vol 65 no 1 pp125ndash140 2015
BioMed Research International 7
[16] F B Costa A R M Barbieri M P J Szabo V N Ramos UPiovezan and M B Labruna ldquoNew records of Rickettsia bellii-infected ticks in Brazilrdquo Brazilian Journal of Veterinary Researchand Animal Science vol 54 no 1 pp 92ndash95 2017
[17] M B Labruna T Whitworth M C Horta et al ldquoRickettsiaspecies infecting Amblyomma cooperi ticks from an area inthe state of Sao Paulo Brazil where Brazilian Spotted Fever isendemicrdquo Journal of Clinical Microbiology vol 42 no 1 pp 90ndash98 2004
[18] K R Macaluso D E Sonenshine S M Ceraul and A F AzadldquoRickettsial infection in Dermacentor variabilis (Acari Ixodi-dae) inhibits transovarial transmission of a second RickettsiardquoJournal of Medical Entomology vol 39 no 6 pp 809ndash813 2002
[19] A Pinter and M B Labruna ldquoIsolation of Rickettsia rickettsiiand Rickettsia bellii in cell culture from the tick Amblyommaaureolatum in Brazilrdquo Annals of the New York Academy ofSciences vol 1078 pp 523ndash529 2006
[20] R De Campos Pacheco M C Horta A Pinter et al ldquoSurvey ofRickettsia spp in the ticks Amblyomma cajennense and Ambly-omma dubitatum in the State of Sao Paulordquo Journal of theBrazilian Society of Tropical Medicine vol 42 no 3 pp 351ndash3532009
[21] G S Sabatini Pesquisa de Carrapatos e Riquetsias no ParqueEstadual da Serra do Mar Nucleo Itutinga ndash Pilloes Sao Paulo[Dissertation thesis] University of Sao Paulo Sao Paulo SP2010
[22] R Pacheco S Rosa L Richtzenhain M P J Szabo and M BLabruna ldquoIsolation of Rickettsia bellii from Amblyomma ovaleand Amblyomma incisum ticks from southern Brazilrdquo RevistaMVZ Cordoba vol 13 no 2 pp 1273ndash1279 2008
[23] M B Labruna R C Pacheco L J Richtzenhain and M P JSzabo ldquoIsolation of Rickettsia rhipicephali and Rickettsia belliifrom Haemaphysalis juxtakochi ticks in the state of Sao PauloBrazilrdquo Applied and Environmental Microbiology vol 73 no 3pp 869ndash873 2007
[24] M C Horta A Pinter T T S Schumaker and M B LabrunaldquoNatural infection transovarial transmission and transstadialsurvival of Rickettsia bellii in the tick Ixodes loricatus (AcariIxodidae) from Brazilrdquo Annals of the New York Academy ofSciences vol 1078 pp 285ndash290 2006
[25] A R M Barbieri Ecologia de carrapatos e rickettsias transmiti-das por carrapatos em uma reserva natural de Cerrado brasileiro[PhD thesis] University of Sao Paulo Sao Paulo SP 2016
[26] F S KrawczakPhD thesis University of Sao Paulo Sao Paulo SPPesquisa de infeccao por riquetsias do grupo da febre maculosaem caes pequenos mamıferos e carrapatos em area endemica enao endemicas nos biomas Pampa eMata Atlantica no estado doRio Grande do Sul [PhD thesis] University of Sao Paulo SaoPaulo SP 2016
[27] M P J Szabo F A Nieri-BastosM G Spolidorio T FMartinsA M Barbieri and M B Labruna ldquoIn vitro isolation fromAmblyomma ovale (Acari Ixodidae) and ecological aspects ofthe Atlantic rainforest Rickettsia the causative agent of a novelspotted fever rickettsiosis in Brazilrdquo Parasitology vol 140 no 6pp 719ndash728 2013
[28] M B Labruna R C Pacheco S Nava P E Brandao L JRichtzenhain and A A Guglielmone ldquoInfection by Rickettsiabellii and Candidatus ldquoRickettsia amblyommiirdquo in Amblyommaneumanni ticks from ArgentinardquoMicrobial Ecology vol 54 no1 pp 126ndash133 2007
[29] J A Hecht M E J Allerdice F S Krawczak M B Labruna CD Paddock and S E Karpathy ldquoDevelopment of a Rickettsia
bellii-specific TaqMan assay targeting the citrate synthase generdquoJournal of Medical Entomology vol 53 no 6 pp 1492ndash14952016
[30] K Poetter PhD thesis Molecular population and evolutionarygenetics of Rickettsia Ohio State University Columbus OHUSA 1989
[31] C D Paddock M E J Allerdice S E Karpathy et al ldquoUniquestrain of Rickettsia parkeri associated with the hard tickDerma-centor parumapertus Neumann in the western United StatesrdquoApplied and Environmental Microbiology vol 83 no 9 ArticleID e03463-16 2017
[32] R N Philip E A Casper and R S Lane ldquoSerotypes of tick-borne spotted fever group Rickettsiae from western CaliforniardquoTheAmerican Journal of Tropical Medicine and Hygiene vol 30no 3 pp 722ndash727 1981
[33] P-E Fournier Y Zhu H Ogata and D Raoult ldquoUse of highlyvariable intergenic spacer sequences for multispacer typing ofRickettsia conorii strainsrdquo Journal of Clinical Microbiology vol42 no 12 pp 5757ndash5766 2004
[34] L Vitorino I M Chelo F Bacellar and L Ze-Ze ldquoRickettsiaephylogeny A multigenic approachrdquo Microbiology vol 153 no1 pp 160ndash168 2007
[35] J D Thompson T J Gibson F Plewniak F Jeanmougin andD G Higgins ldquoThe CLUSTAL X windows interface flexiblestrategies for multiple sequence alignment aided by qualityanalysis toolsrdquoNucleic Acids Research vol 25 no 24 pp 4876ndash4882 1997
[36] K Tamura G Stecher D Peterson A Filipski and S KumarldquoMEGA6 Molecular Evolutionary Genetics Analysis version60rdquoMolecular Biology and Evolution vol 30 no 12 pp 2725ndash2729 2013
[37] T H Ogden andM S Rosenberg ldquoHow should gaps be treatedin parsimony A comparison of approaches using simulationrdquoMolecular Phylogenetics and Evolution vol 42 no 3 pp 817ndash826 2007
[38] J P Huelsenbeck and F Ronquist ldquoMrBayes Bayesian inferenceof phylogenetic treesrdquo Bioinformatics vol 17 no 8 pp 754-7552001
[39] R W Emmons D V Dondero B C Nelson and R S LaneldquoEcology of tick-borne agents in Californiardquo The AmericanJournal of Tropical Medicine and Hygiene vol 30 no 1 pp 239ndash252 1981
[40] R C Pacheco M C Horta J Moraes-Filho A C Ataliba APinter and M B Labruna ldquoRickettsial infection in capybaras(Hyrochoerus hydrochaeris) from Sao Paulo Brazil Serologicalevidence for infection by Rickettsia bellii and Rickettsia parkerirdquoBiomedica vol 27 no 3 pp 364ndash371 2008
[41] M G Coelho V do Nascimento Ramos J E Limongi etal ldquoSerologic evidence of the exposure of small mammals tospotted-fever Rickettsia and Rickettsia bellii in Minas GeraisBrazilrdquoThe Journal of Infection in Developing Countries vol 10no 3 pp 275ndash282 2016
[42] RHu K EHyland andOliver ldquoA review on the use of ixodiph-agus wasps (Hymenoptera Encyrtidae) as natural enemies forthe control of ticks (Acari Ixodidae)rdquo Systematic and AppliedAcarology vol 3 pp 19ndash28 1998
[43] A J O Lopes J R S Nascimento-Junior C G Silva A PPrado M B Labruna and L M Costa-Junior ldquoParasitismby ixodiphagus wasps (Hymenoptera Encyrtidae) in Rhipi-cephalus sanguineus and Amblyomma ticks (Acari Ixodidae) inthree regions of Brazilrdquo Journal of Economic Entomology vol105 no 6 pp 1979ndash1981 2012
8 BioMed Research International
[44] M Bohacsova O Mediannikov M Kazimirova D Raoult andZ Sekeyova ldquoArsenophonus nasoniae and rickettsiae infectionof Ixodes ricinus due to parasitic wasp Ixodiphagus hookerirdquoPLoS ONE vol 11 no 2 Article ID e0149950 2016
[45] HOgata B La Scola S Audic et al ldquoGenome sequence ofRick-ettsia bellii illuminates the role of amoebae in gene exchangesbetween intracellular pathogensrdquo PLoS Genetics vol 2 no 5pp 733ndash744 2006
[46] S J Perlman M S Hunter and E Zchori-Fein ldquoThe emergingdiversity of Rickettsiardquo Proceedings of the Royal Society B Bio-logical Science vol 273 no 1598 pp 2097ndash2106 2006
[47] H Klompen and D Grimaldi ldquoFirst mesozoic record of aparasitiform mite A larval argasid tick in Cretaceous Amber(Acari Ixodida Argasidae)rdquoAnnals of the Entomological Societyof America vol 94 no 1 pp 10ndash15 2001
[48] D A Grimaldi M S Engel and P C Nascimbene ldquoFossilifer-ous cretaceous amber fromMyanmar (Burma) Its rediscoverybiotic diversity and paleontological significancerdquo AmericanMuseum Novitates vol 3361 pp 1ndash71 2002
[49] S Nava A A Guglielmone and A J Mangold ldquoAn overview ofsystematics and evolution of ticksrdquo Frontiers in Bioscience vol14 no 8 pp 2857ndash2877 2009
[50] A A Guglielmone D A Apanaskevich A Estrada-Pena R GRobbins T N Petney and I G Horak The Hard Ticks of theWorld (Acari Ixodida Ixodidae) vol 1 Springer Netherlandspress Dordrecht 1st edition 2014
Hindawiwwwhindawicom
International Journal of
Volume 2018
Zoology
Hindawiwwwhindawicom Volume 2018
Anatomy Research International
PeptidesInternational Journal of
Hindawiwwwhindawicom Volume 2018
Hindawiwwwhindawicom Volume 2018
Journal of Parasitology Research
GenomicsInternational Journal of
Hindawiwwwhindawicom Volume 2018
Hindawi Publishing Corporation httpwwwhindawicom Volume 2013Hindawiwwwhindawicom
The Scientific World Journal
Volume 2018
Hindawiwwwhindawicom Volume 2018
BioinformaticsAdvances in
Marine BiologyJournal of
Hindawiwwwhindawicom Volume 2018
Hindawiwwwhindawicom Volume 2018
Neuroscience Journal
Hindawiwwwhindawicom Volume 2018
BioMed Research International
Cell BiologyInternational Journal of
Hindawiwwwhindawicom Volume 2018
Hindawiwwwhindawicom Volume 2018
Biochemistry Research International
ArchaeaHindawiwwwhindawicom Volume 2018
Hindawiwwwhindawicom Volume 2018
Genetics Research International
Hindawiwwwhindawicom Volume 2018
Advances in
Virolog y Stem Cells International
Hindawiwwwhindawicom Volume 2018
Hindawiwwwhindawicom Volume 2018
Enzyme Research
Hindawiwwwhindawicom Volume 2018
International Journal of
MicrobiologyHindawiwwwhindawicom
Nucleic AcidsJournal of
Volume 2018
Submit your manuscripts atwwwhindawicom
4 BioMed Research International
Table 1 Rickettsia bellii isolates from South and North America used in the present study
Numberlowast Isolate name Tick host Geographic origin Reference(1) Mogi Amblyomma aureolatum Mogi das Cruzes-SP Brazil [19](2) Ad-MG Amblyomma dubitatum Guarda-Mor-MG Brazil [16](3) Ad-CORD-SP A dubitatum Cordeiropolis -SP Brazil [20](4) Ad-25-INT A dubitatum Ribeirao Grande-SP Brazil [20](5) PNSM Amblyomma incisum Cubatao-SP Brazil [21](6) AO Amblyomma ovale Ribeirao Grande-SP Brazil [22](7) HJ4 Haemaphysalis juxtakochi Ribeirao Grande-SP Brazil [23](8) IL-Mogi Ixodes loricatus Mogi das Cruzes-SP Brazil [24](9) Ap GSV 136 Amblyomma parvum Chapada Gaucha-MG Brazil [25](10) IL-RS1 I loricatus Derrubadas-RS Brazil [26](11) RB-CL I loricatus Cerro Largo-RS Brazil [13](12) HJ1 H juxtakochi Sao Paulo-SP Brazil [23](13) Ap-MS Amblyomma pseudoconcolor Corumba-MS Brazil [16](14) P-10 A ovale Peruıbe-SP Brazil [27](15) An4 Amblyomma neumanni Dean Funes-Cordoba Argentina [28](16) 369-C Dermacentor variabilis Washington Co Arkansas USA [5](17) CA13-1 Dermacentor variabilis Yolo Co California USA [29](18) CA13-9 D variabilis Yolo Co California USA [29](19) CA13-17 D variabilis Yolo Co California USA [29](20) Putah Creek D variabilis Solano Co California USA [29](21) Yolo D variabilis Yolo Co California USA [29](22) Stevenson Bridge D variabilis Yolo Co California USA [29](23) OSU 83-1223 D variabilis Knox County Ohio USA [30](24) TX15-1 Dermacentor parumapertus Brewster County Texas USA [31](25) OSU 83-117 D variabilis Licking County Ohio USA [30](26) OSU 85-1299 D variabilis Morrow County Ohio USA [30](27) OSU 83-452 D variabilis Coshocton County Ohio USA [30](28) Skull Valley D parumapertus Tooele County Utah USA [31](29) OSU 85-389 D variabilis Ohio USA [30](30) CA-459 Haemaphysalis leporispalustris Mendocino Co California USA [32]lowastThese numbers are represented in Figure 1 as the geographical location of each isolate
a large genome size with high coding capacity in contrastto the reduced genome size with low coding capacity of thepathogenic rickettsiae [4 45]
It has been proposed that the R bellii group divergedprior to the division between the SFG and the TG forming abasal group that also includes various herbivorous arthropodsymbionts [1 46] While the genus Rickettsia was consideredto be approximately 150 million years old the divergenceof the R bellii group was estimated to have occurred muchmore recently around 50 million years ago [1] Analysesof the oldest tick fossils from the Cretaceous indicate thatextant tick genera (Amblyomma Ornithodoros)were soundlyestablished around 100 million years ago [47ndash49] Fromthese data it can be inferred that R bellii likely radiatedwith its principal hosts approximately 50 million years agoWhile ticks and rickettsiae are distributed in all continentsit is noteworthy that under natural conditions and withouthuman interference tick species from the NewWorld do notoccur in the Old World and vice versa the only exceptionsare a few tick species that are associated with transcontinental
marine birds [50] Indeed this scenario has accounted forthe presence of R bellii-infected ticks restricted to the NewWorld regardless of the ability of this bacterium to infecta vast array of tick genera and species On the other handwhether R bellii originated with ticks in North or SouthAmerica remains obscure since our phylogenetic analysesdid not show any evidence for greater genetic divergenceof R bellii in any of the two continents Further analysesencompassing more R bellii isolates from different tickgenera and species encompassing more molecular markersshould provide cues for the origin and radiation of R bellii inthe NewWorld
5 Conclusion
Phylogeographical analysis of 30 strains (15 from NorthAmerica and 15 from South America) isolated from 13species of 4 genera shows a clear differentiation betweenmost of the North and South American isolates indicatinggeographic isolation between isolates of these two continents
BioMed Research International 5
Table2Prim
erpairs
used
fora
mplificatio
nof
rickettsialgenes
orintergenicregion
sinthep
resent
study
Prim
erpair
Target
Forw
ardprim
er(51015840to
31015840)
Reversep
rimer
(51015840to
31015840)
Ann
ealin
gtemperature
(∘ C)
Amplicon
size(nt)
Reference
(1)
mppA-purC
GCA
ATTA
TCGGTC
CGAAT
GTT
TCAT
TTAT
TTGTC
TCAAAAT
TCA
45to
551
160
[33]
(2)
nusG
-rplK
CAGTT
GCA
ATAT
TGGTA
AAG
CACA
GCA
GCT
GGAAT
TATC
AAG
TT45
to551
270
[33]
(3)
murG-RC0
563
GAAG
AAAAG
AAG
GGCA
TAAG
CTA
CAAG
CTGAAAG
TAAAAAC
ATTC
C40
to521
293
[33]
(4)
ntrY-rpsU
AGCT
GCT
GTT
GCT
AAAG
TAAAAA
CAAG
AAG
CAGCA
AGAAG
ACAG
A52
to641
363
[33]
(5)
dksA-xerC
TCCC
ATAG
GTA
ATTT
AGGTG
TTTC
TACT
ACCG
CATA
TCCA
ATTA
AAAA
45to
551
416
[33]
(6)
spo0J-a
bcT1
AAAG
ATTT
GGAAG
AAT
TAGAC
TTGAT
TTTG
CTTA
AAC
CAAC
CATT
TCA
45to
551
259
[33]
(7)
fabZ
-lpxD
TGTT
AGGAT
CGAT
TTTA
AGTA
CTCT
ATCT
TGGAT
TGGCA
TAGAC
AAT
CTAT
TA45
to551
195
[33]
(8)
RC1137-tlc-5
CGGGAT
AAC
GCC
GAG
TAAT
AAT
GCC
GCT
CTGAAT
TTGTT
T45
to551
264
[33]
(9)
RC0230-RC0
231
TGCA
CCCG
CCTA
AAAC
TAAC
ATGGTC
GGCC
GTA
GAAAAA
45to
551
232
[33]
(10)
groE
S-RC
0970
CTTG
CATC
GGCT
TTTC
TTTT
AGCT
TTGAG
CTGAT
GGGCT
A45
to551
215
[33]
(11)
rrf-p
yrH
GAG
CTTT
CTCC
ATCT
TTTC
TTG
AAAG
GGGAAT
ATAC
GAC
AAT
TGAG
45to
551
238
[33]
(12)
tRNA
Gly-tR
NA
Tyr
AGCT
TGGAAG
GCT
GGAAC
TCAT
CCTT
CTCC
CTCC
ACCA
CT45
to551
148
[33]
(13)
pal-R
C1201
TGCA
AGCA
CACA
TAAT
GCA
ATC
AAAAT
CGAT
TCCT
CTTT
TCC
45to
551
216
[33]
(14)
atpA
ATCA
AGCG
TTGCA
CAGAT
AGGGAAG
TGCC
GTA
AGTG
AAC
C58
Unk
nownlowast
[1]
(15)
rpmE-tRNA
fMet
TTCC
GGAAAT
GTA
GTA
AAT
CAAT
CTC
AGGTT
ATGAG
CCTG
ACGA
54144
[33]
(16)
RC1027-xthA2
GGTA
TGTA
AAT
GAG
CCTT
ATCA
ATAC
TTC
AGTA
GTA
TAAG
TAGCT
CCTG
CTGTC
54351
[33]
(17)
gltA
GTC
TACT
GCT
TCGTG
TAGAT
CAAC
GGCT
GAC
CTAT
AGAAT
ATTT
ATAAG
AC54
408
[25]
(18)
coxA
ACAG
CCGTT
GAT
ATGGCT
ACA
TATT
CCAAC
CGGCA
AAAG
58Unk
nownlowast
[1]
(19)
coxA
GGTG
CTCC
TGAT
ATGGCA
TTCA
TATT
CCAG
CCGGCA
AAAG
58Unk
nownlowast
[1]
(20)
atpA
ATCA
AGCG
TTGCA
CAGAT
AGCG
ACTT
ACCG
AAAT
ACCG
AC56
449
[134]
lowastDatan
otgivenin
theo
riginalpu
blication1teste
din
gradient
PCR
6 BioMed Research International
Additionally this analysis separated isolates of R bellii by thespecies of tick fromwhich these were isolated indicating thatthe isolates could have coevolved with their tick vectors overtime
Disclosure
The findings and conclusions are those of the authors anddo not necessarily reflect the views of the US Departmentof Health and Human Services or the official position of theCenters for Disease Control and Prevention
Conflicts of Interest
The authors declare that there are no conflicts of interestregarding the publication of this manuscript
Acknowledgments
This work received financial support from the Sao PauloResearch Foundation (FAPESP Grant 201510060-6)
Supplementary Materials
An identity matrix table of the 2269-nucleotide alignmentused for the phylogeny if R bellii is available in thesupplemental material for this article (Table S1) The finalalignment comprising 2269 nucleotides was concatenated inthe following order rpmE -tRNAfmet (414-nt) gltA (357-nt) coxA (898-nt) atpA (449-nt) and RC1027 -xthA2 (151-nt) and it is also available in the supplementary material(Rbellii final alignmentfas) Two additional phylogenetictrees are also available in the supplementary material (RbelliiGTR treepdf and Rbellii HKY treepdf) Table S1 identitymatrix of a 2269-nucleotide alignment of the 30 Rickettsiabellii isolates from Brazil (BRA) Argentina (ARG) and theUnited States (USA) used in the present study SupplementaryFigure 1 molecular phylogenetic analysis of 30 isolates ofRickettsia bellii from North and South America A total of2269 aligned nucleotide sites of 3 protein coding genes (gltAatpA and coxA) and 2 intergenic regions ( rpmE-tRNAfmet and RC1027- xthA2) were concatenated and subjectedto Bayesian analysis A total of 10000000 generations wererun using the GTRmodel with a sample frequency of 10000The analysis was run with 3 heated chains and the first1000000 generations were discarded as burn-in Numbers atnodes are support values derived from posterior probabilityThe scale bar is in units of expected substitutions per siteSupplementary Figure 2 molecular phylogenetic analysis of30 isolates of Rickettsia bellii fromNorth and South AmericaA total of 2269 aligned nucleotide sites of 3 protein codinggenes (gltA atpA and coxA) and 2 intergenic regions(rpmE-tRNA fmet and RC1027- xthA2) were concatenatedand subjected to Bayesian analysis A total of 10000000generations were run using the HKY85 model with a samplefrequency of 10000 The analysis was run with 3 heatedchains and the first 1000000 generations were discarded asburn-in Numbers at nodes are support values derived from
posterior probability The scale bar is in units of expectedsubstitutions per site (Supplementary Materials)
References
[1] L A Weinert J H Werren A Aebi G N Stone and F MJiggins ldquoEvolution and diversity of Rickettsia bacteriardquo BMCBiology vol 7 article no 6 2009
[2] P Parola C D Paddock C Socolovschi et al ldquoUpdate on tick-borne rickettsioses around the world a geographic approachrdquoClinical Microbiology Reviews vol 26 no 4 pp 657ndash702 2013
[3] D R Stothard J B Clark and P A Fuerst ldquoAncestral diver-gence of Rickettsia bellii from the spotted fever and typhusgroups of Rickettsia and antiquity of the genus Rickettsiardquo Inter-national Journal of Systematic Bacteriology vol 44 no 4 pp798ndash804 1994
[4] V Merhej and D Raoult ldquoRickettsial evolution in the light ofcomparative genomicsrdquo Biological Reviews vol 86 no 2 pp379ndash405 2011
[5] R N Philip E A Casper and R L Anacker ldquoRickettsia belliisp nov A tick-borne Rickettsia widely distributed in theUnited States that is distinct from the spotted fever and typhusbiogroupsrdquo International Journal of Systematic Bacteriology vol33 no 1 pp 94ndash106 1983
[6] M B Labruna S Mattar V and S Nava ldquoRickettsioses inLatin America Caribbean Spain and Portugalrdquo Revista MVZCordoba vol 16 no 2 pp 2435ndash2457 2011
[7] M Ogrzewalska I Literak J M Cardenas-CallirgosM Capekand M B Labruna ldquoRickettsia bellii in ticks Amblyommavarium Koch 1844 from birds in Perurdquo Ticks and Tick-borneDiseases vol 3 no 4 pp 254ndash256 2012
[8] M Ogrzewalska D G Saraiva J Moraes-Filho et al ldquoEpidemi-ology of Brazilian spotted fever in the Atlantic Forest state ofSao Paulo Brazilrdquo Parasitology vol 139 no 10 pp 1283ndash13002012
[9] J Miranda and S Mattar ldquoMolecular detection of Rickettsiabellii and Rickettsia sp strain Colombianensi in ticks fromCordoba Colombiardquo Ticks and Tick-borne Diseases vol 5 no2 pp 208ndash212 2014
[10] M Andoh A Sakata A Takano et al ldquoDetection of Rickettsiaand Ehrlichia spp In ticks associated with exotic reptiles andamphibians imported into Japanrdquo PLoS ONE vol 10 no 7Article ID e0133700 2015
[11] M Ogrzewalska I Literak M Capek et al ldquoBacteria of thegenus Rickettsia in ticks (Acari Ixodidae) collected from birdsin Costa Ricardquo Ticks and Tick-borne Diseases vol 6 no 4 pp478ndash482 2015
[12] A R M Barbieri L Romero and M B Labruna ldquoRickettsiabellii infecting Amblyomma sabanerae ticks in El SalvadorrdquoPathogens and Global Health vol 106 no 3 pp 188-189 2012
[13] F S Krawczak L C Binder C S Oliveira et al ldquoEcology of atick-borne spotted fever in southern Brazilrdquo Experimental andApplied Acarology vol 70 no 2 pp 219ndash229 2016
[14] D McIntosh R A Bezerra H R Luz et al ldquoDetectionof Rickettsia bellii and Rickettsia amblyommii in Amblyommalongirostre (Acari Ixodidae) fromBahia state Northeast BrazilrdquoBrazilian Journal of Microbiology vol 46 no 3 pp 879ndash8832015
[15] H S Soares A R M Barbieri T F Martins et al ldquoTicks andrickettsial infection in thewildlife of two regions of the BrazilianAmazonrdquo Experimental and Applied Acarology vol 65 no 1 pp125ndash140 2015
BioMed Research International 7
[16] F B Costa A R M Barbieri M P J Szabo V N Ramos UPiovezan and M B Labruna ldquoNew records of Rickettsia bellii-infected ticks in Brazilrdquo Brazilian Journal of Veterinary Researchand Animal Science vol 54 no 1 pp 92ndash95 2017
[17] M B Labruna T Whitworth M C Horta et al ldquoRickettsiaspecies infecting Amblyomma cooperi ticks from an area inthe state of Sao Paulo Brazil where Brazilian Spotted Fever isendemicrdquo Journal of Clinical Microbiology vol 42 no 1 pp 90ndash98 2004
[18] K R Macaluso D E Sonenshine S M Ceraul and A F AzadldquoRickettsial infection in Dermacentor variabilis (Acari Ixodi-dae) inhibits transovarial transmission of a second RickettsiardquoJournal of Medical Entomology vol 39 no 6 pp 809ndash813 2002
[19] A Pinter and M B Labruna ldquoIsolation of Rickettsia rickettsiiand Rickettsia bellii in cell culture from the tick Amblyommaaureolatum in Brazilrdquo Annals of the New York Academy ofSciences vol 1078 pp 523ndash529 2006
[20] R De Campos Pacheco M C Horta A Pinter et al ldquoSurvey ofRickettsia spp in the ticks Amblyomma cajennense and Ambly-omma dubitatum in the State of Sao Paulordquo Journal of theBrazilian Society of Tropical Medicine vol 42 no 3 pp 351ndash3532009
[21] G S Sabatini Pesquisa de Carrapatos e Riquetsias no ParqueEstadual da Serra do Mar Nucleo Itutinga ndash Pilloes Sao Paulo[Dissertation thesis] University of Sao Paulo Sao Paulo SP2010
[22] R Pacheco S Rosa L Richtzenhain M P J Szabo and M BLabruna ldquoIsolation of Rickettsia bellii from Amblyomma ovaleand Amblyomma incisum ticks from southern Brazilrdquo RevistaMVZ Cordoba vol 13 no 2 pp 1273ndash1279 2008
[23] M B Labruna R C Pacheco L J Richtzenhain and M P JSzabo ldquoIsolation of Rickettsia rhipicephali and Rickettsia belliifrom Haemaphysalis juxtakochi ticks in the state of Sao PauloBrazilrdquo Applied and Environmental Microbiology vol 73 no 3pp 869ndash873 2007
[24] M C Horta A Pinter T T S Schumaker and M B LabrunaldquoNatural infection transovarial transmission and transstadialsurvival of Rickettsia bellii in the tick Ixodes loricatus (AcariIxodidae) from Brazilrdquo Annals of the New York Academy ofSciences vol 1078 pp 285ndash290 2006
[25] A R M Barbieri Ecologia de carrapatos e rickettsias transmiti-das por carrapatos em uma reserva natural de Cerrado brasileiro[PhD thesis] University of Sao Paulo Sao Paulo SP 2016
[26] F S KrawczakPhD thesis University of Sao Paulo Sao Paulo SPPesquisa de infeccao por riquetsias do grupo da febre maculosaem caes pequenos mamıferos e carrapatos em area endemica enao endemicas nos biomas Pampa eMata Atlantica no estado doRio Grande do Sul [PhD thesis] University of Sao Paulo SaoPaulo SP 2016
[27] M P J Szabo F A Nieri-BastosM G Spolidorio T FMartinsA M Barbieri and M B Labruna ldquoIn vitro isolation fromAmblyomma ovale (Acari Ixodidae) and ecological aspects ofthe Atlantic rainforest Rickettsia the causative agent of a novelspotted fever rickettsiosis in Brazilrdquo Parasitology vol 140 no 6pp 719ndash728 2013
[28] M B Labruna R C Pacheco S Nava P E Brandao L JRichtzenhain and A A Guglielmone ldquoInfection by Rickettsiabellii and Candidatus ldquoRickettsia amblyommiirdquo in Amblyommaneumanni ticks from ArgentinardquoMicrobial Ecology vol 54 no1 pp 126ndash133 2007
[29] J A Hecht M E J Allerdice F S Krawczak M B Labruna CD Paddock and S E Karpathy ldquoDevelopment of a Rickettsia
bellii-specific TaqMan assay targeting the citrate synthase generdquoJournal of Medical Entomology vol 53 no 6 pp 1492ndash14952016
[30] K Poetter PhD thesis Molecular population and evolutionarygenetics of Rickettsia Ohio State University Columbus OHUSA 1989
[31] C D Paddock M E J Allerdice S E Karpathy et al ldquoUniquestrain of Rickettsia parkeri associated with the hard tickDerma-centor parumapertus Neumann in the western United StatesrdquoApplied and Environmental Microbiology vol 83 no 9 ArticleID e03463-16 2017
[32] R N Philip E A Casper and R S Lane ldquoSerotypes of tick-borne spotted fever group Rickettsiae from western CaliforniardquoTheAmerican Journal of Tropical Medicine and Hygiene vol 30no 3 pp 722ndash727 1981
[33] P-E Fournier Y Zhu H Ogata and D Raoult ldquoUse of highlyvariable intergenic spacer sequences for multispacer typing ofRickettsia conorii strainsrdquo Journal of Clinical Microbiology vol42 no 12 pp 5757ndash5766 2004
[34] L Vitorino I M Chelo F Bacellar and L Ze-Ze ldquoRickettsiaephylogeny A multigenic approachrdquo Microbiology vol 153 no1 pp 160ndash168 2007
[35] J D Thompson T J Gibson F Plewniak F Jeanmougin andD G Higgins ldquoThe CLUSTAL X windows interface flexiblestrategies for multiple sequence alignment aided by qualityanalysis toolsrdquoNucleic Acids Research vol 25 no 24 pp 4876ndash4882 1997
[36] K Tamura G Stecher D Peterson A Filipski and S KumarldquoMEGA6 Molecular Evolutionary Genetics Analysis version60rdquoMolecular Biology and Evolution vol 30 no 12 pp 2725ndash2729 2013
[37] T H Ogden andM S Rosenberg ldquoHow should gaps be treatedin parsimony A comparison of approaches using simulationrdquoMolecular Phylogenetics and Evolution vol 42 no 3 pp 817ndash826 2007
[38] J P Huelsenbeck and F Ronquist ldquoMrBayes Bayesian inferenceof phylogenetic treesrdquo Bioinformatics vol 17 no 8 pp 754-7552001
[39] R W Emmons D V Dondero B C Nelson and R S LaneldquoEcology of tick-borne agents in Californiardquo The AmericanJournal of Tropical Medicine and Hygiene vol 30 no 1 pp 239ndash252 1981
[40] R C Pacheco M C Horta J Moraes-Filho A C Ataliba APinter and M B Labruna ldquoRickettsial infection in capybaras(Hyrochoerus hydrochaeris) from Sao Paulo Brazil Serologicalevidence for infection by Rickettsia bellii and Rickettsia parkerirdquoBiomedica vol 27 no 3 pp 364ndash371 2008
[41] M G Coelho V do Nascimento Ramos J E Limongi etal ldquoSerologic evidence of the exposure of small mammals tospotted-fever Rickettsia and Rickettsia bellii in Minas GeraisBrazilrdquoThe Journal of Infection in Developing Countries vol 10no 3 pp 275ndash282 2016
[42] RHu K EHyland andOliver ldquoA review on the use of ixodiph-agus wasps (Hymenoptera Encyrtidae) as natural enemies forthe control of ticks (Acari Ixodidae)rdquo Systematic and AppliedAcarology vol 3 pp 19ndash28 1998
[43] A J O Lopes J R S Nascimento-Junior C G Silva A PPrado M B Labruna and L M Costa-Junior ldquoParasitismby ixodiphagus wasps (Hymenoptera Encyrtidae) in Rhipi-cephalus sanguineus and Amblyomma ticks (Acari Ixodidae) inthree regions of Brazilrdquo Journal of Economic Entomology vol105 no 6 pp 1979ndash1981 2012
8 BioMed Research International
[44] M Bohacsova O Mediannikov M Kazimirova D Raoult andZ Sekeyova ldquoArsenophonus nasoniae and rickettsiae infectionof Ixodes ricinus due to parasitic wasp Ixodiphagus hookerirdquoPLoS ONE vol 11 no 2 Article ID e0149950 2016
[45] HOgata B La Scola S Audic et al ldquoGenome sequence ofRick-ettsia bellii illuminates the role of amoebae in gene exchangesbetween intracellular pathogensrdquo PLoS Genetics vol 2 no 5pp 733ndash744 2006
[46] S J Perlman M S Hunter and E Zchori-Fein ldquoThe emergingdiversity of Rickettsiardquo Proceedings of the Royal Society B Bio-logical Science vol 273 no 1598 pp 2097ndash2106 2006
[47] H Klompen and D Grimaldi ldquoFirst mesozoic record of aparasitiform mite A larval argasid tick in Cretaceous Amber(Acari Ixodida Argasidae)rdquoAnnals of the Entomological Societyof America vol 94 no 1 pp 10ndash15 2001
[48] D A Grimaldi M S Engel and P C Nascimbene ldquoFossilifer-ous cretaceous amber fromMyanmar (Burma) Its rediscoverybiotic diversity and paleontological significancerdquo AmericanMuseum Novitates vol 3361 pp 1ndash71 2002
[49] S Nava A A Guglielmone and A J Mangold ldquoAn overview ofsystematics and evolution of ticksrdquo Frontiers in Bioscience vol14 no 8 pp 2857ndash2877 2009
[50] A A Guglielmone D A Apanaskevich A Estrada-Pena R GRobbins T N Petney and I G Horak The Hard Ticks of theWorld (Acari Ixodida Ixodidae) vol 1 Springer Netherlandspress Dordrecht 1st edition 2014
Hindawiwwwhindawicom
International Journal of
Volume 2018
Zoology
Hindawiwwwhindawicom Volume 2018
Anatomy Research International
PeptidesInternational Journal of
Hindawiwwwhindawicom Volume 2018
Hindawiwwwhindawicom Volume 2018
Journal of Parasitology Research
GenomicsInternational Journal of
Hindawiwwwhindawicom Volume 2018
Hindawi Publishing Corporation httpwwwhindawicom Volume 2013Hindawiwwwhindawicom
The Scientific World Journal
Volume 2018
Hindawiwwwhindawicom Volume 2018
BioinformaticsAdvances in
Marine BiologyJournal of
Hindawiwwwhindawicom Volume 2018
Hindawiwwwhindawicom Volume 2018
Neuroscience Journal
Hindawiwwwhindawicom Volume 2018
BioMed Research International
Cell BiologyInternational Journal of
Hindawiwwwhindawicom Volume 2018
Hindawiwwwhindawicom Volume 2018
Biochemistry Research International
ArchaeaHindawiwwwhindawicom Volume 2018
Hindawiwwwhindawicom Volume 2018
Genetics Research International
Hindawiwwwhindawicom Volume 2018
Advances in
Virolog y Stem Cells International
Hindawiwwwhindawicom Volume 2018
Hindawiwwwhindawicom Volume 2018
Enzyme Research
Hindawiwwwhindawicom Volume 2018
International Journal of
MicrobiologyHindawiwwwhindawicom
Nucleic AcidsJournal of
Volume 2018
Submit your manuscripts atwwwhindawicom
BioMed Research International 5
Table2Prim
erpairs
used
fora
mplificatio
nof
rickettsialgenes
orintergenicregion
sinthep
resent
study
Prim
erpair
Target
Forw
ardprim
er(51015840to
31015840)
Reversep
rimer
(51015840to
31015840)
Ann
ealin
gtemperature
(∘ C)
Amplicon
size(nt)
Reference
(1)
mppA-purC
GCA
ATTA
TCGGTC
CGAAT
GTT
TCAT
TTAT
TTGTC
TCAAAAT
TCA
45to
551
160
[33]
(2)
nusG
-rplK
CAGTT
GCA
ATAT
TGGTA
AAG
CACA
GCA
GCT
GGAAT
TATC
AAG
TT45
to551
270
[33]
(3)
murG-RC0
563
GAAG
AAAAG
AAG
GGCA
TAAG
CTA
CAAG
CTGAAAG
TAAAAAC
ATTC
C40
to521
293
[33]
(4)
ntrY-rpsU
AGCT
GCT
GTT
GCT
AAAG
TAAAAA
CAAG
AAG
CAGCA
AGAAG
ACAG
A52
to641
363
[33]
(5)
dksA-xerC
TCCC
ATAG
GTA
ATTT
AGGTG
TTTC
TACT
ACCG
CATA
TCCA
ATTA
AAAA
45to
551
416
[33]
(6)
spo0J-a
bcT1
AAAG
ATTT
GGAAG
AAT
TAGAC
TTGAT
TTTG
CTTA
AAC
CAAC
CATT
TCA
45to
551
259
[33]
(7)
fabZ
-lpxD
TGTT
AGGAT
CGAT
TTTA
AGTA
CTCT
ATCT
TGGAT
TGGCA
TAGAC
AAT
CTAT
TA45
to551
195
[33]
(8)
RC1137-tlc-5
CGGGAT
AAC
GCC
GAG
TAAT
AAT
GCC
GCT
CTGAAT
TTGTT
T45
to551
264
[33]
(9)
RC0230-RC0
231
TGCA
CCCG
CCTA
AAAC
TAAC
ATGGTC
GGCC
GTA
GAAAAA
45to
551
232
[33]
(10)
groE
S-RC
0970
CTTG
CATC
GGCT
TTTC
TTTT
AGCT
TTGAG
CTGAT
GGGCT
A45
to551
215
[33]
(11)
rrf-p
yrH
GAG
CTTT
CTCC
ATCT
TTTC
TTG
AAAG
GGGAAT
ATAC
GAC
AAT
TGAG
45to
551
238
[33]
(12)
tRNA
Gly-tR
NA
Tyr
AGCT
TGGAAG
GCT
GGAAC
TCAT
CCTT
CTCC
CTCC
ACCA
CT45
to551
148
[33]
(13)
pal-R
C1201
TGCA
AGCA
CACA
TAAT
GCA
ATC
AAAAT
CGAT
TCCT
CTTT
TCC
45to
551
216
[33]
(14)
atpA
ATCA
AGCG
TTGCA
CAGAT
AGGGAAG
TGCC
GTA
AGTG
AAC
C58
Unk
nownlowast
[1]
(15)
rpmE-tRNA
fMet
TTCC
GGAAAT
GTA
GTA
AAT
CAAT
CTC
AGGTT
ATGAG
CCTG
ACGA
54144
[33]
(16)
RC1027-xthA2
GGTA
TGTA
AAT
GAG
CCTT
ATCA
ATAC
TTC
AGTA
GTA
TAAG
TAGCT
CCTG
CTGTC
54351
[33]
(17)
gltA
GTC
TACT
GCT
TCGTG
TAGAT
CAAC
GGCT
GAC
CTAT
AGAAT
ATTT
ATAAG
AC54
408
[25]
(18)
coxA
ACAG
CCGTT
GAT
ATGGCT
ACA
TATT
CCAAC
CGGCA
AAAG
58Unk
nownlowast
[1]
(19)
coxA
GGTG
CTCC
TGAT
ATGGCA
TTCA
TATT
CCAG
CCGGCA
AAAG
58Unk
nownlowast
[1]
(20)
atpA
ATCA
AGCG
TTGCA
CAGAT
AGCG
ACTT
ACCG
AAAT
ACCG
AC56
449
[134]
lowastDatan
otgivenin
theo
riginalpu
blication1teste
din
gradient
PCR
6 BioMed Research International
Additionally this analysis separated isolates of R bellii by thespecies of tick fromwhich these were isolated indicating thatthe isolates could have coevolved with their tick vectors overtime
Disclosure
The findings and conclusions are those of the authors anddo not necessarily reflect the views of the US Departmentof Health and Human Services or the official position of theCenters for Disease Control and Prevention
Conflicts of Interest
The authors declare that there are no conflicts of interestregarding the publication of this manuscript
Acknowledgments
This work received financial support from the Sao PauloResearch Foundation (FAPESP Grant 201510060-6)
Supplementary Materials
An identity matrix table of the 2269-nucleotide alignmentused for the phylogeny if R bellii is available in thesupplemental material for this article (Table S1) The finalalignment comprising 2269 nucleotides was concatenated inthe following order rpmE -tRNAfmet (414-nt) gltA (357-nt) coxA (898-nt) atpA (449-nt) and RC1027 -xthA2 (151-nt) and it is also available in the supplementary material(Rbellii final alignmentfas) Two additional phylogenetictrees are also available in the supplementary material (RbelliiGTR treepdf and Rbellii HKY treepdf) Table S1 identitymatrix of a 2269-nucleotide alignment of the 30 Rickettsiabellii isolates from Brazil (BRA) Argentina (ARG) and theUnited States (USA) used in the present study SupplementaryFigure 1 molecular phylogenetic analysis of 30 isolates ofRickettsia bellii from North and South America A total of2269 aligned nucleotide sites of 3 protein coding genes (gltAatpA and coxA) and 2 intergenic regions ( rpmE-tRNAfmet and RC1027- xthA2) were concatenated and subjectedto Bayesian analysis A total of 10000000 generations wererun using the GTRmodel with a sample frequency of 10000The analysis was run with 3 heated chains and the first1000000 generations were discarded as burn-in Numbers atnodes are support values derived from posterior probabilityThe scale bar is in units of expected substitutions per siteSupplementary Figure 2 molecular phylogenetic analysis of30 isolates of Rickettsia bellii fromNorth and South AmericaA total of 2269 aligned nucleotide sites of 3 protein codinggenes (gltA atpA and coxA) and 2 intergenic regions(rpmE-tRNA fmet and RC1027- xthA2) were concatenatedand subjected to Bayesian analysis A total of 10000000generations were run using the HKY85 model with a samplefrequency of 10000 The analysis was run with 3 heatedchains and the first 1000000 generations were discarded asburn-in Numbers at nodes are support values derived from
posterior probability The scale bar is in units of expectedsubstitutions per site (Supplementary Materials)
References
[1] L A Weinert J H Werren A Aebi G N Stone and F MJiggins ldquoEvolution and diversity of Rickettsia bacteriardquo BMCBiology vol 7 article no 6 2009
[2] P Parola C D Paddock C Socolovschi et al ldquoUpdate on tick-borne rickettsioses around the world a geographic approachrdquoClinical Microbiology Reviews vol 26 no 4 pp 657ndash702 2013
[3] D R Stothard J B Clark and P A Fuerst ldquoAncestral diver-gence of Rickettsia bellii from the spotted fever and typhusgroups of Rickettsia and antiquity of the genus Rickettsiardquo Inter-national Journal of Systematic Bacteriology vol 44 no 4 pp798ndash804 1994
[4] V Merhej and D Raoult ldquoRickettsial evolution in the light ofcomparative genomicsrdquo Biological Reviews vol 86 no 2 pp379ndash405 2011
[5] R N Philip E A Casper and R L Anacker ldquoRickettsia belliisp nov A tick-borne Rickettsia widely distributed in theUnited States that is distinct from the spotted fever and typhusbiogroupsrdquo International Journal of Systematic Bacteriology vol33 no 1 pp 94ndash106 1983
[6] M B Labruna S Mattar V and S Nava ldquoRickettsioses inLatin America Caribbean Spain and Portugalrdquo Revista MVZCordoba vol 16 no 2 pp 2435ndash2457 2011
[7] M Ogrzewalska I Literak J M Cardenas-CallirgosM Capekand M B Labruna ldquoRickettsia bellii in ticks Amblyommavarium Koch 1844 from birds in Perurdquo Ticks and Tick-borneDiseases vol 3 no 4 pp 254ndash256 2012
[8] M Ogrzewalska D G Saraiva J Moraes-Filho et al ldquoEpidemi-ology of Brazilian spotted fever in the Atlantic Forest state ofSao Paulo Brazilrdquo Parasitology vol 139 no 10 pp 1283ndash13002012
[9] J Miranda and S Mattar ldquoMolecular detection of Rickettsiabellii and Rickettsia sp strain Colombianensi in ticks fromCordoba Colombiardquo Ticks and Tick-borne Diseases vol 5 no2 pp 208ndash212 2014
[10] M Andoh A Sakata A Takano et al ldquoDetection of Rickettsiaand Ehrlichia spp In ticks associated with exotic reptiles andamphibians imported into Japanrdquo PLoS ONE vol 10 no 7Article ID e0133700 2015
[11] M Ogrzewalska I Literak M Capek et al ldquoBacteria of thegenus Rickettsia in ticks (Acari Ixodidae) collected from birdsin Costa Ricardquo Ticks and Tick-borne Diseases vol 6 no 4 pp478ndash482 2015
[12] A R M Barbieri L Romero and M B Labruna ldquoRickettsiabellii infecting Amblyomma sabanerae ticks in El SalvadorrdquoPathogens and Global Health vol 106 no 3 pp 188-189 2012
[13] F S Krawczak L C Binder C S Oliveira et al ldquoEcology of atick-borne spotted fever in southern Brazilrdquo Experimental andApplied Acarology vol 70 no 2 pp 219ndash229 2016
[14] D McIntosh R A Bezerra H R Luz et al ldquoDetectionof Rickettsia bellii and Rickettsia amblyommii in Amblyommalongirostre (Acari Ixodidae) fromBahia state Northeast BrazilrdquoBrazilian Journal of Microbiology vol 46 no 3 pp 879ndash8832015
[15] H S Soares A R M Barbieri T F Martins et al ldquoTicks andrickettsial infection in thewildlife of two regions of the BrazilianAmazonrdquo Experimental and Applied Acarology vol 65 no 1 pp125ndash140 2015
BioMed Research International 7
[16] F B Costa A R M Barbieri M P J Szabo V N Ramos UPiovezan and M B Labruna ldquoNew records of Rickettsia bellii-infected ticks in Brazilrdquo Brazilian Journal of Veterinary Researchand Animal Science vol 54 no 1 pp 92ndash95 2017
[17] M B Labruna T Whitworth M C Horta et al ldquoRickettsiaspecies infecting Amblyomma cooperi ticks from an area inthe state of Sao Paulo Brazil where Brazilian Spotted Fever isendemicrdquo Journal of Clinical Microbiology vol 42 no 1 pp 90ndash98 2004
[18] K R Macaluso D E Sonenshine S M Ceraul and A F AzadldquoRickettsial infection in Dermacentor variabilis (Acari Ixodi-dae) inhibits transovarial transmission of a second RickettsiardquoJournal of Medical Entomology vol 39 no 6 pp 809ndash813 2002
[19] A Pinter and M B Labruna ldquoIsolation of Rickettsia rickettsiiand Rickettsia bellii in cell culture from the tick Amblyommaaureolatum in Brazilrdquo Annals of the New York Academy ofSciences vol 1078 pp 523ndash529 2006
[20] R De Campos Pacheco M C Horta A Pinter et al ldquoSurvey ofRickettsia spp in the ticks Amblyomma cajennense and Ambly-omma dubitatum in the State of Sao Paulordquo Journal of theBrazilian Society of Tropical Medicine vol 42 no 3 pp 351ndash3532009
[21] G S Sabatini Pesquisa de Carrapatos e Riquetsias no ParqueEstadual da Serra do Mar Nucleo Itutinga ndash Pilloes Sao Paulo[Dissertation thesis] University of Sao Paulo Sao Paulo SP2010
[22] R Pacheco S Rosa L Richtzenhain M P J Szabo and M BLabruna ldquoIsolation of Rickettsia bellii from Amblyomma ovaleand Amblyomma incisum ticks from southern Brazilrdquo RevistaMVZ Cordoba vol 13 no 2 pp 1273ndash1279 2008
[23] M B Labruna R C Pacheco L J Richtzenhain and M P JSzabo ldquoIsolation of Rickettsia rhipicephali and Rickettsia belliifrom Haemaphysalis juxtakochi ticks in the state of Sao PauloBrazilrdquo Applied and Environmental Microbiology vol 73 no 3pp 869ndash873 2007
[24] M C Horta A Pinter T T S Schumaker and M B LabrunaldquoNatural infection transovarial transmission and transstadialsurvival of Rickettsia bellii in the tick Ixodes loricatus (AcariIxodidae) from Brazilrdquo Annals of the New York Academy ofSciences vol 1078 pp 285ndash290 2006
[25] A R M Barbieri Ecologia de carrapatos e rickettsias transmiti-das por carrapatos em uma reserva natural de Cerrado brasileiro[PhD thesis] University of Sao Paulo Sao Paulo SP 2016
[26] F S KrawczakPhD thesis University of Sao Paulo Sao Paulo SPPesquisa de infeccao por riquetsias do grupo da febre maculosaem caes pequenos mamıferos e carrapatos em area endemica enao endemicas nos biomas Pampa eMata Atlantica no estado doRio Grande do Sul [PhD thesis] University of Sao Paulo SaoPaulo SP 2016
[27] M P J Szabo F A Nieri-BastosM G Spolidorio T FMartinsA M Barbieri and M B Labruna ldquoIn vitro isolation fromAmblyomma ovale (Acari Ixodidae) and ecological aspects ofthe Atlantic rainforest Rickettsia the causative agent of a novelspotted fever rickettsiosis in Brazilrdquo Parasitology vol 140 no 6pp 719ndash728 2013
[28] M B Labruna R C Pacheco S Nava P E Brandao L JRichtzenhain and A A Guglielmone ldquoInfection by Rickettsiabellii and Candidatus ldquoRickettsia amblyommiirdquo in Amblyommaneumanni ticks from ArgentinardquoMicrobial Ecology vol 54 no1 pp 126ndash133 2007
[29] J A Hecht M E J Allerdice F S Krawczak M B Labruna CD Paddock and S E Karpathy ldquoDevelopment of a Rickettsia
bellii-specific TaqMan assay targeting the citrate synthase generdquoJournal of Medical Entomology vol 53 no 6 pp 1492ndash14952016
[30] K Poetter PhD thesis Molecular population and evolutionarygenetics of Rickettsia Ohio State University Columbus OHUSA 1989
[31] C D Paddock M E J Allerdice S E Karpathy et al ldquoUniquestrain of Rickettsia parkeri associated with the hard tickDerma-centor parumapertus Neumann in the western United StatesrdquoApplied and Environmental Microbiology vol 83 no 9 ArticleID e03463-16 2017
[32] R N Philip E A Casper and R S Lane ldquoSerotypes of tick-borne spotted fever group Rickettsiae from western CaliforniardquoTheAmerican Journal of Tropical Medicine and Hygiene vol 30no 3 pp 722ndash727 1981
[33] P-E Fournier Y Zhu H Ogata and D Raoult ldquoUse of highlyvariable intergenic spacer sequences for multispacer typing ofRickettsia conorii strainsrdquo Journal of Clinical Microbiology vol42 no 12 pp 5757ndash5766 2004
[34] L Vitorino I M Chelo F Bacellar and L Ze-Ze ldquoRickettsiaephylogeny A multigenic approachrdquo Microbiology vol 153 no1 pp 160ndash168 2007
[35] J D Thompson T J Gibson F Plewniak F Jeanmougin andD G Higgins ldquoThe CLUSTAL X windows interface flexiblestrategies for multiple sequence alignment aided by qualityanalysis toolsrdquoNucleic Acids Research vol 25 no 24 pp 4876ndash4882 1997
[36] K Tamura G Stecher D Peterson A Filipski and S KumarldquoMEGA6 Molecular Evolutionary Genetics Analysis version60rdquoMolecular Biology and Evolution vol 30 no 12 pp 2725ndash2729 2013
[37] T H Ogden andM S Rosenberg ldquoHow should gaps be treatedin parsimony A comparison of approaches using simulationrdquoMolecular Phylogenetics and Evolution vol 42 no 3 pp 817ndash826 2007
[38] J P Huelsenbeck and F Ronquist ldquoMrBayes Bayesian inferenceof phylogenetic treesrdquo Bioinformatics vol 17 no 8 pp 754-7552001
[39] R W Emmons D V Dondero B C Nelson and R S LaneldquoEcology of tick-borne agents in Californiardquo The AmericanJournal of Tropical Medicine and Hygiene vol 30 no 1 pp 239ndash252 1981
[40] R C Pacheco M C Horta J Moraes-Filho A C Ataliba APinter and M B Labruna ldquoRickettsial infection in capybaras(Hyrochoerus hydrochaeris) from Sao Paulo Brazil Serologicalevidence for infection by Rickettsia bellii and Rickettsia parkerirdquoBiomedica vol 27 no 3 pp 364ndash371 2008
[41] M G Coelho V do Nascimento Ramos J E Limongi etal ldquoSerologic evidence of the exposure of small mammals tospotted-fever Rickettsia and Rickettsia bellii in Minas GeraisBrazilrdquoThe Journal of Infection in Developing Countries vol 10no 3 pp 275ndash282 2016
[42] RHu K EHyland andOliver ldquoA review on the use of ixodiph-agus wasps (Hymenoptera Encyrtidae) as natural enemies forthe control of ticks (Acari Ixodidae)rdquo Systematic and AppliedAcarology vol 3 pp 19ndash28 1998
[43] A J O Lopes J R S Nascimento-Junior C G Silva A PPrado M B Labruna and L M Costa-Junior ldquoParasitismby ixodiphagus wasps (Hymenoptera Encyrtidae) in Rhipi-cephalus sanguineus and Amblyomma ticks (Acari Ixodidae) inthree regions of Brazilrdquo Journal of Economic Entomology vol105 no 6 pp 1979ndash1981 2012
8 BioMed Research International
[44] M Bohacsova O Mediannikov M Kazimirova D Raoult andZ Sekeyova ldquoArsenophonus nasoniae and rickettsiae infectionof Ixodes ricinus due to parasitic wasp Ixodiphagus hookerirdquoPLoS ONE vol 11 no 2 Article ID e0149950 2016
[45] HOgata B La Scola S Audic et al ldquoGenome sequence ofRick-ettsia bellii illuminates the role of amoebae in gene exchangesbetween intracellular pathogensrdquo PLoS Genetics vol 2 no 5pp 733ndash744 2006
[46] S J Perlman M S Hunter and E Zchori-Fein ldquoThe emergingdiversity of Rickettsiardquo Proceedings of the Royal Society B Bio-logical Science vol 273 no 1598 pp 2097ndash2106 2006
[47] H Klompen and D Grimaldi ldquoFirst mesozoic record of aparasitiform mite A larval argasid tick in Cretaceous Amber(Acari Ixodida Argasidae)rdquoAnnals of the Entomological Societyof America vol 94 no 1 pp 10ndash15 2001
[48] D A Grimaldi M S Engel and P C Nascimbene ldquoFossilifer-ous cretaceous amber fromMyanmar (Burma) Its rediscoverybiotic diversity and paleontological significancerdquo AmericanMuseum Novitates vol 3361 pp 1ndash71 2002
[49] S Nava A A Guglielmone and A J Mangold ldquoAn overview ofsystematics and evolution of ticksrdquo Frontiers in Bioscience vol14 no 8 pp 2857ndash2877 2009
[50] A A Guglielmone D A Apanaskevich A Estrada-Pena R GRobbins T N Petney and I G Horak The Hard Ticks of theWorld (Acari Ixodida Ixodidae) vol 1 Springer Netherlandspress Dordrecht 1st edition 2014
Hindawiwwwhindawicom
International Journal of
Volume 2018
Zoology
Hindawiwwwhindawicom Volume 2018
Anatomy Research International
PeptidesInternational Journal of
Hindawiwwwhindawicom Volume 2018
Hindawiwwwhindawicom Volume 2018
Journal of Parasitology Research
GenomicsInternational Journal of
Hindawiwwwhindawicom Volume 2018
Hindawi Publishing Corporation httpwwwhindawicom Volume 2013Hindawiwwwhindawicom
The Scientific World Journal
Volume 2018
Hindawiwwwhindawicom Volume 2018
BioinformaticsAdvances in
Marine BiologyJournal of
Hindawiwwwhindawicom Volume 2018
Hindawiwwwhindawicom Volume 2018
Neuroscience Journal
Hindawiwwwhindawicom Volume 2018
BioMed Research International
Cell BiologyInternational Journal of
Hindawiwwwhindawicom Volume 2018
Hindawiwwwhindawicom Volume 2018
Biochemistry Research International
ArchaeaHindawiwwwhindawicom Volume 2018
Hindawiwwwhindawicom Volume 2018
Genetics Research International
Hindawiwwwhindawicom Volume 2018
Advances in
Virolog y Stem Cells International
Hindawiwwwhindawicom Volume 2018
Hindawiwwwhindawicom Volume 2018
Enzyme Research
Hindawiwwwhindawicom Volume 2018
International Journal of
MicrobiologyHindawiwwwhindawicom
Nucleic AcidsJournal of
Volume 2018
Submit your manuscripts atwwwhindawicom
6 BioMed Research International
Additionally this analysis separated isolates of R bellii by thespecies of tick fromwhich these were isolated indicating thatthe isolates could have coevolved with their tick vectors overtime
Disclosure
The findings and conclusions are those of the authors anddo not necessarily reflect the views of the US Departmentof Health and Human Services or the official position of theCenters for Disease Control and Prevention
Conflicts of Interest
The authors declare that there are no conflicts of interestregarding the publication of this manuscript
Acknowledgments
This work received financial support from the Sao PauloResearch Foundation (FAPESP Grant 201510060-6)
Supplementary Materials
An identity matrix table of the 2269-nucleotide alignmentused for the phylogeny if R bellii is available in thesupplemental material for this article (Table S1) The finalalignment comprising 2269 nucleotides was concatenated inthe following order rpmE -tRNAfmet (414-nt) gltA (357-nt) coxA (898-nt) atpA (449-nt) and RC1027 -xthA2 (151-nt) and it is also available in the supplementary material(Rbellii final alignmentfas) Two additional phylogenetictrees are also available in the supplementary material (RbelliiGTR treepdf and Rbellii HKY treepdf) Table S1 identitymatrix of a 2269-nucleotide alignment of the 30 Rickettsiabellii isolates from Brazil (BRA) Argentina (ARG) and theUnited States (USA) used in the present study SupplementaryFigure 1 molecular phylogenetic analysis of 30 isolates ofRickettsia bellii from North and South America A total of2269 aligned nucleotide sites of 3 protein coding genes (gltAatpA and coxA) and 2 intergenic regions ( rpmE-tRNAfmet and RC1027- xthA2) were concatenated and subjectedto Bayesian analysis A total of 10000000 generations wererun using the GTRmodel with a sample frequency of 10000The analysis was run with 3 heated chains and the first1000000 generations were discarded as burn-in Numbers atnodes are support values derived from posterior probabilityThe scale bar is in units of expected substitutions per siteSupplementary Figure 2 molecular phylogenetic analysis of30 isolates of Rickettsia bellii fromNorth and South AmericaA total of 2269 aligned nucleotide sites of 3 protein codinggenes (gltA atpA and coxA) and 2 intergenic regions(rpmE-tRNA fmet and RC1027- xthA2) were concatenatedand subjected to Bayesian analysis A total of 10000000generations were run using the HKY85 model with a samplefrequency of 10000 The analysis was run with 3 heatedchains and the first 1000000 generations were discarded asburn-in Numbers at nodes are support values derived from
posterior probability The scale bar is in units of expectedsubstitutions per site (Supplementary Materials)
References
[1] L A Weinert J H Werren A Aebi G N Stone and F MJiggins ldquoEvolution and diversity of Rickettsia bacteriardquo BMCBiology vol 7 article no 6 2009
[2] P Parola C D Paddock C Socolovschi et al ldquoUpdate on tick-borne rickettsioses around the world a geographic approachrdquoClinical Microbiology Reviews vol 26 no 4 pp 657ndash702 2013
[3] D R Stothard J B Clark and P A Fuerst ldquoAncestral diver-gence of Rickettsia bellii from the spotted fever and typhusgroups of Rickettsia and antiquity of the genus Rickettsiardquo Inter-national Journal of Systematic Bacteriology vol 44 no 4 pp798ndash804 1994
[4] V Merhej and D Raoult ldquoRickettsial evolution in the light ofcomparative genomicsrdquo Biological Reviews vol 86 no 2 pp379ndash405 2011
[5] R N Philip E A Casper and R L Anacker ldquoRickettsia belliisp nov A tick-borne Rickettsia widely distributed in theUnited States that is distinct from the spotted fever and typhusbiogroupsrdquo International Journal of Systematic Bacteriology vol33 no 1 pp 94ndash106 1983
[6] M B Labruna S Mattar V and S Nava ldquoRickettsioses inLatin America Caribbean Spain and Portugalrdquo Revista MVZCordoba vol 16 no 2 pp 2435ndash2457 2011
[7] M Ogrzewalska I Literak J M Cardenas-CallirgosM Capekand M B Labruna ldquoRickettsia bellii in ticks Amblyommavarium Koch 1844 from birds in Perurdquo Ticks and Tick-borneDiseases vol 3 no 4 pp 254ndash256 2012
[8] M Ogrzewalska D G Saraiva J Moraes-Filho et al ldquoEpidemi-ology of Brazilian spotted fever in the Atlantic Forest state ofSao Paulo Brazilrdquo Parasitology vol 139 no 10 pp 1283ndash13002012
[9] J Miranda and S Mattar ldquoMolecular detection of Rickettsiabellii and Rickettsia sp strain Colombianensi in ticks fromCordoba Colombiardquo Ticks and Tick-borne Diseases vol 5 no2 pp 208ndash212 2014
[10] M Andoh A Sakata A Takano et al ldquoDetection of Rickettsiaand Ehrlichia spp In ticks associated with exotic reptiles andamphibians imported into Japanrdquo PLoS ONE vol 10 no 7Article ID e0133700 2015
[11] M Ogrzewalska I Literak M Capek et al ldquoBacteria of thegenus Rickettsia in ticks (Acari Ixodidae) collected from birdsin Costa Ricardquo Ticks and Tick-borne Diseases vol 6 no 4 pp478ndash482 2015
[12] A R M Barbieri L Romero and M B Labruna ldquoRickettsiabellii infecting Amblyomma sabanerae ticks in El SalvadorrdquoPathogens and Global Health vol 106 no 3 pp 188-189 2012
[13] F S Krawczak L C Binder C S Oliveira et al ldquoEcology of atick-borne spotted fever in southern Brazilrdquo Experimental andApplied Acarology vol 70 no 2 pp 219ndash229 2016
[14] D McIntosh R A Bezerra H R Luz et al ldquoDetectionof Rickettsia bellii and Rickettsia amblyommii in Amblyommalongirostre (Acari Ixodidae) fromBahia state Northeast BrazilrdquoBrazilian Journal of Microbiology vol 46 no 3 pp 879ndash8832015
[15] H S Soares A R M Barbieri T F Martins et al ldquoTicks andrickettsial infection in thewildlife of two regions of the BrazilianAmazonrdquo Experimental and Applied Acarology vol 65 no 1 pp125ndash140 2015
BioMed Research International 7
[16] F B Costa A R M Barbieri M P J Szabo V N Ramos UPiovezan and M B Labruna ldquoNew records of Rickettsia bellii-infected ticks in Brazilrdquo Brazilian Journal of Veterinary Researchand Animal Science vol 54 no 1 pp 92ndash95 2017
[17] M B Labruna T Whitworth M C Horta et al ldquoRickettsiaspecies infecting Amblyomma cooperi ticks from an area inthe state of Sao Paulo Brazil where Brazilian Spotted Fever isendemicrdquo Journal of Clinical Microbiology vol 42 no 1 pp 90ndash98 2004
[18] K R Macaluso D E Sonenshine S M Ceraul and A F AzadldquoRickettsial infection in Dermacentor variabilis (Acari Ixodi-dae) inhibits transovarial transmission of a second RickettsiardquoJournal of Medical Entomology vol 39 no 6 pp 809ndash813 2002
[19] A Pinter and M B Labruna ldquoIsolation of Rickettsia rickettsiiand Rickettsia bellii in cell culture from the tick Amblyommaaureolatum in Brazilrdquo Annals of the New York Academy ofSciences vol 1078 pp 523ndash529 2006
[20] R De Campos Pacheco M C Horta A Pinter et al ldquoSurvey ofRickettsia spp in the ticks Amblyomma cajennense and Ambly-omma dubitatum in the State of Sao Paulordquo Journal of theBrazilian Society of Tropical Medicine vol 42 no 3 pp 351ndash3532009
[21] G S Sabatini Pesquisa de Carrapatos e Riquetsias no ParqueEstadual da Serra do Mar Nucleo Itutinga ndash Pilloes Sao Paulo[Dissertation thesis] University of Sao Paulo Sao Paulo SP2010
[22] R Pacheco S Rosa L Richtzenhain M P J Szabo and M BLabruna ldquoIsolation of Rickettsia bellii from Amblyomma ovaleand Amblyomma incisum ticks from southern Brazilrdquo RevistaMVZ Cordoba vol 13 no 2 pp 1273ndash1279 2008
[23] M B Labruna R C Pacheco L J Richtzenhain and M P JSzabo ldquoIsolation of Rickettsia rhipicephali and Rickettsia belliifrom Haemaphysalis juxtakochi ticks in the state of Sao PauloBrazilrdquo Applied and Environmental Microbiology vol 73 no 3pp 869ndash873 2007
[24] M C Horta A Pinter T T S Schumaker and M B LabrunaldquoNatural infection transovarial transmission and transstadialsurvival of Rickettsia bellii in the tick Ixodes loricatus (AcariIxodidae) from Brazilrdquo Annals of the New York Academy ofSciences vol 1078 pp 285ndash290 2006
[25] A R M Barbieri Ecologia de carrapatos e rickettsias transmiti-das por carrapatos em uma reserva natural de Cerrado brasileiro[PhD thesis] University of Sao Paulo Sao Paulo SP 2016
[26] F S KrawczakPhD thesis University of Sao Paulo Sao Paulo SPPesquisa de infeccao por riquetsias do grupo da febre maculosaem caes pequenos mamıferos e carrapatos em area endemica enao endemicas nos biomas Pampa eMata Atlantica no estado doRio Grande do Sul [PhD thesis] University of Sao Paulo SaoPaulo SP 2016
[27] M P J Szabo F A Nieri-BastosM G Spolidorio T FMartinsA M Barbieri and M B Labruna ldquoIn vitro isolation fromAmblyomma ovale (Acari Ixodidae) and ecological aspects ofthe Atlantic rainforest Rickettsia the causative agent of a novelspotted fever rickettsiosis in Brazilrdquo Parasitology vol 140 no 6pp 719ndash728 2013
[28] M B Labruna R C Pacheco S Nava P E Brandao L JRichtzenhain and A A Guglielmone ldquoInfection by Rickettsiabellii and Candidatus ldquoRickettsia amblyommiirdquo in Amblyommaneumanni ticks from ArgentinardquoMicrobial Ecology vol 54 no1 pp 126ndash133 2007
[29] J A Hecht M E J Allerdice F S Krawczak M B Labruna CD Paddock and S E Karpathy ldquoDevelopment of a Rickettsia
bellii-specific TaqMan assay targeting the citrate synthase generdquoJournal of Medical Entomology vol 53 no 6 pp 1492ndash14952016
[30] K Poetter PhD thesis Molecular population and evolutionarygenetics of Rickettsia Ohio State University Columbus OHUSA 1989
[31] C D Paddock M E J Allerdice S E Karpathy et al ldquoUniquestrain of Rickettsia parkeri associated with the hard tickDerma-centor parumapertus Neumann in the western United StatesrdquoApplied and Environmental Microbiology vol 83 no 9 ArticleID e03463-16 2017
[32] R N Philip E A Casper and R S Lane ldquoSerotypes of tick-borne spotted fever group Rickettsiae from western CaliforniardquoTheAmerican Journal of Tropical Medicine and Hygiene vol 30no 3 pp 722ndash727 1981
[33] P-E Fournier Y Zhu H Ogata and D Raoult ldquoUse of highlyvariable intergenic spacer sequences for multispacer typing ofRickettsia conorii strainsrdquo Journal of Clinical Microbiology vol42 no 12 pp 5757ndash5766 2004
[34] L Vitorino I M Chelo F Bacellar and L Ze-Ze ldquoRickettsiaephylogeny A multigenic approachrdquo Microbiology vol 153 no1 pp 160ndash168 2007
[35] J D Thompson T J Gibson F Plewniak F Jeanmougin andD G Higgins ldquoThe CLUSTAL X windows interface flexiblestrategies for multiple sequence alignment aided by qualityanalysis toolsrdquoNucleic Acids Research vol 25 no 24 pp 4876ndash4882 1997
[36] K Tamura G Stecher D Peterson A Filipski and S KumarldquoMEGA6 Molecular Evolutionary Genetics Analysis version60rdquoMolecular Biology and Evolution vol 30 no 12 pp 2725ndash2729 2013
[37] T H Ogden andM S Rosenberg ldquoHow should gaps be treatedin parsimony A comparison of approaches using simulationrdquoMolecular Phylogenetics and Evolution vol 42 no 3 pp 817ndash826 2007
[38] J P Huelsenbeck and F Ronquist ldquoMrBayes Bayesian inferenceof phylogenetic treesrdquo Bioinformatics vol 17 no 8 pp 754-7552001
[39] R W Emmons D V Dondero B C Nelson and R S LaneldquoEcology of tick-borne agents in Californiardquo The AmericanJournal of Tropical Medicine and Hygiene vol 30 no 1 pp 239ndash252 1981
[40] R C Pacheco M C Horta J Moraes-Filho A C Ataliba APinter and M B Labruna ldquoRickettsial infection in capybaras(Hyrochoerus hydrochaeris) from Sao Paulo Brazil Serologicalevidence for infection by Rickettsia bellii and Rickettsia parkerirdquoBiomedica vol 27 no 3 pp 364ndash371 2008
[41] M G Coelho V do Nascimento Ramos J E Limongi etal ldquoSerologic evidence of the exposure of small mammals tospotted-fever Rickettsia and Rickettsia bellii in Minas GeraisBrazilrdquoThe Journal of Infection in Developing Countries vol 10no 3 pp 275ndash282 2016
[42] RHu K EHyland andOliver ldquoA review on the use of ixodiph-agus wasps (Hymenoptera Encyrtidae) as natural enemies forthe control of ticks (Acari Ixodidae)rdquo Systematic and AppliedAcarology vol 3 pp 19ndash28 1998
[43] A J O Lopes J R S Nascimento-Junior C G Silva A PPrado M B Labruna and L M Costa-Junior ldquoParasitismby ixodiphagus wasps (Hymenoptera Encyrtidae) in Rhipi-cephalus sanguineus and Amblyomma ticks (Acari Ixodidae) inthree regions of Brazilrdquo Journal of Economic Entomology vol105 no 6 pp 1979ndash1981 2012
8 BioMed Research International
[44] M Bohacsova O Mediannikov M Kazimirova D Raoult andZ Sekeyova ldquoArsenophonus nasoniae and rickettsiae infectionof Ixodes ricinus due to parasitic wasp Ixodiphagus hookerirdquoPLoS ONE vol 11 no 2 Article ID e0149950 2016
[45] HOgata B La Scola S Audic et al ldquoGenome sequence ofRick-ettsia bellii illuminates the role of amoebae in gene exchangesbetween intracellular pathogensrdquo PLoS Genetics vol 2 no 5pp 733ndash744 2006
[46] S J Perlman M S Hunter and E Zchori-Fein ldquoThe emergingdiversity of Rickettsiardquo Proceedings of the Royal Society B Bio-logical Science vol 273 no 1598 pp 2097ndash2106 2006
[47] H Klompen and D Grimaldi ldquoFirst mesozoic record of aparasitiform mite A larval argasid tick in Cretaceous Amber(Acari Ixodida Argasidae)rdquoAnnals of the Entomological Societyof America vol 94 no 1 pp 10ndash15 2001
[48] D A Grimaldi M S Engel and P C Nascimbene ldquoFossilifer-ous cretaceous amber fromMyanmar (Burma) Its rediscoverybiotic diversity and paleontological significancerdquo AmericanMuseum Novitates vol 3361 pp 1ndash71 2002
[49] S Nava A A Guglielmone and A J Mangold ldquoAn overview ofsystematics and evolution of ticksrdquo Frontiers in Bioscience vol14 no 8 pp 2857ndash2877 2009
[50] A A Guglielmone D A Apanaskevich A Estrada-Pena R GRobbins T N Petney and I G Horak The Hard Ticks of theWorld (Acari Ixodida Ixodidae) vol 1 Springer Netherlandspress Dordrecht 1st edition 2014
Hindawiwwwhindawicom
International Journal of
Volume 2018
Zoology
Hindawiwwwhindawicom Volume 2018
Anatomy Research International
PeptidesInternational Journal of
Hindawiwwwhindawicom Volume 2018
Hindawiwwwhindawicom Volume 2018
Journal of Parasitology Research
GenomicsInternational Journal of
Hindawiwwwhindawicom Volume 2018
Hindawi Publishing Corporation httpwwwhindawicom Volume 2013Hindawiwwwhindawicom
The Scientific World Journal
Volume 2018
Hindawiwwwhindawicom Volume 2018
BioinformaticsAdvances in
Marine BiologyJournal of
Hindawiwwwhindawicom Volume 2018
Hindawiwwwhindawicom Volume 2018
Neuroscience Journal
Hindawiwwwhindawicom Volume 2018
BioMed Research International
Cell BiologyInternational Journal of
Hindawiwwwhindawicom Volume 2018
Hindawiwwwhindawicom Volume 2018
Biochemistry Research International
ArchaeaHindawiwwwhindawicom Volume 2018
Hindawiwwwhindawicom Volume 2018
Genetics Research International
Hindawiwwwhindawicom Volume 2018
Advances in
Virolog y Stem Cells International
Hindawiwwwhindawicom Volume 2018
Hindawiwwwhindawicom Volume 2018
Enzyme Research
Hindawiwwwhindawicom Volume 2018
International Journal of
MicrobiologyHindawiwwwhindawicom
Nucleic AcidsJournal of
Volume 2018
Submit your manuscripts atwwwhindawicom
BioMed Research International 7
[16] F B Costa A R M Barbieri M P J Szabo V N Ramos UPiovezan and M B Labruna ldquoNew records of Rickettsia bellii-infected ticks in Brazilrdquo Brazilian Journal of Veterinary Researchand Animal Science vol 54 no 1 pp 92ndash95 2017
[17] M B Labruna T Whitworth M C Horta et al ldquoRickettsiaspecies infecting Amblyomma cooperi ticks from an area inthe state of Sao Paulo Brazil where Brazilian Spotted Fever isendemicrdquo Journal of Clinical Microbiology vol 42 no 1 pp 90ndash98 2004
[18] K R Macaluso D E Sonenshine S M Ceraul and A F AzadldquoRickettsial infection in Dermacentor variabilis (Acari Ixodi-dae) inhibits transovarial transmission of a second RickettsiardquoJournal of Medical Entomology vol 39 no 6 pp 809ndash813 2002
[19] A Pinter and M B Labruna ldquoIsolation of Rickettsia rickettsiiand Rickettsia bellii in cell culture from the tick Amblyommaaureolatum in Brazilrdquo Annals of the New York Academy ofSciences vol 1078 pp 523ndash529 2006
[20] R De Campos Pacheco M C Horta A Pinter et al ldquoSurvey ofRickettsia spp in the ticks Amblyomma cajennense and Ambly-omma dubitatum in the State of Sao Paulordquo Journal of theBrazilian Society of Tropical Medicine vol 42 no 3 pp 351ndash3532009
[21] G S Sabatini Pesquisa de Carrapatos e Riquetsias no ParqueEstadual da Serra do Mar Nucleo Itutinga ndash Pilloes Sao Paulo[Dissertation thesis] University of Sao Paulo Sao Paulo SP2010
[22] R Pacheco S Rosa L Richtzenhain M P J Szabo and M BLabruna ldquoIsolation of Rickettsia bellii from Amblyomma ovaleand Amblyomma incisum ticks from southern Brazilrdquo RevistaMVZ Cordoba vol 13 no 2 pp 1273ndash1279 2008
[23] M B Labruna R C Pacheco L J Richtzenhain and M P JSzabo ldquoIsolation of Rickettsia rhipicephali and Rickettsia belliifrom Haemaphysalis juxtakochi ticks in the state of Sao PauloBrazilrdquo Applied and Environmental Microbiology vol 73 no 3pp 869ndash873 2007
[24] M C Horta A Pinter T T S Schumaker and M B LabrunaldquoNatural infection transovarial transmission and transstadialsurvival of Rickettsia bellii in the tick Ixodes loricatus (AcariIxodidae) from Brazilrdquo Annals of the New York Academy ofSciences vol 1078 pp 285ndash290 2006
[25] A R M Barbieri Ecologia de carrapatos e rickettsias transmiti-das por carrapatos em uma reserva natural de Cerrado brasileiro[PhD thesis] University of Sao Paulo Sao Paulo SP 2016
[26] F S KrawczakPhD thesis University of Sao Paulo Sao Paulo SPPesquisa de infeccao por riquetsias do grupo da febre maculosaem caes pequenos mamıferos e carrapatos em area endemica enao endemicas nos biomas Pampa eMata Atlantica no estado doRio Grande do Sul [PhD thesis] University of Sao Paulo SaoPaulo SP 2016
[27] M P J Szabo F A Nieri-BastosM G Spolidorio T FMartinsA M Barbieri and M B Labruna ldquoIn vitro isolation fromAmblyomma ovale (Acari Ixodidae) and ecological aspects ofthe Atlantic rainforest Rickettsia the causative agent of a novelspotted fever rickettsiosis in Brazilrdquo Parasitology vol 140 no 6pp 719ndash728 2013
[28] M B Labruna R C Pacheco S Nava P E Brandao L JRichtzenhain and A A Guglielmone ldquoInfection by Rickettsiabellii and Candidatus ldquoRickettsia amblyommiirdquo in Amblyommaneumanni ticks from ArgentinardquoMicrobial Ecology vol 54 no1 pp 126ndash133 2007
[29] J A Hecht M E J Allerdice F S Krawczak M B Labruna CD Paddock and S E Karpathy ldquoDevelopment of a Rickettsia
bellii-specific TaqMan assay targeting the citrate synthase generdquoJournal of Medical Entomology vol 53 no 6 pp 1492ndash14952016
[30] K Poetter PhD thesis Molecular population and evolutionarygenetics of Rickettsia Ohio State University Columbus OHUSA 1989
[31] C D Paddock M E J Allerdice S E Karpathy et al ldquoUniquestrain of Rickettsia parkeri associated with the hard tickDerma-centor parumapertus Neumann in the western United StatesrdquoApplied and Environmental Microbiology vol 83 no 9 ArticleID e03463-16 2017
[32] R N Philip E A Casper and R S Lane ldquoSerotypes of tick-borne spotted fever group Rickettsiae from western CaliforniardquoTheAmerican Journal of Tropical Medicine and Hygiene vol 30no 3 pp 722ndash727 1981
[33] P-E Fournier Y Zhu H Ogata and D Raoult ldquoUse of highlyvariable intergenic spacer sequences for multispacer typing ofRickettsia conorii strainsrdquo Journal of Clinical Microbiology vol42 no 12 pp 5757ndash5766 2004
[34] L Vitorino I M Chelo F Bacellar and L Ze-Ze ldquoRickettsiaephylogeny A multigenic approachrdquo Microbiology vol 153 no1 pp 160ndash168 2007
[35] J D Thompson T J Gibson F Plewniak F Jeanmougin andD G Higgins ldquoThe CLUSTAL X windows interface flexiblestrategies for multiple sequence alignment aided by qualityanalysis toolsrdquoNucleic Acids Research vol 25 no 24 pp 4876ndash4882 1997
[36] K Tamura G Stecher D Peterson A Filipski and S KumarldquoMEGA6 Molecular Evolutionary Genetics Analysis version60rdquoMolecular Biology and Evolution vol 30 no 12 pp 2725ndash2729 2013
[37] T H Ogden andM S Rosenberg ldquoHow should gaps be treatedin parsimony A comparison of approaches using simulationrdquoMolecular Phylogenetics and Evolution vol 42 no 3 pp 817ndash826 2007
[38] J P Huelsenbeck and F Ronquist ldquoMrBayes Bayesian inferenceof phylogenetic treesrdquo Bioinformatics vol 17 no 8 pp 754-7552001
[39] R W Emmons D V Dondero B C Nelson and R S LaneldquoEcology of tick-borne agents in Californiardquo The AmericanJournal of Tropical Medicine and Hygiene vol 30 no 1 pp 239ndash252 1981
[40] R C Pacheco M C Horta J Moraes-Filho A C Ataliba APinter and M B Labruna ldquoRickettsial infection in capybaras(Hyrochoerus hydrochaeris) from Sao Paulo Brazil Serologicalevidence for infection by Rickettsia bellii and Rickettsia parkerirdquoBiomedica vol 27 no 3 pp 364ndash371 2008
[41] M G Coelho V do Nascimento Ramos J E Limongi etal ldquoSerologic evidence of the exposure of small mammals tospotted-fever Rickettsia and Rickettsia bellii in Minas GeraisBrazilrdquoThe Journal of Infection in Developing Countries vol 10no 3 pp 275ndash282 2016
[42] RHu K EHyland andOliver ldquoA review on the use of ixodiph-agus wasps (Hymenoptera Encyrtidae) as natural enemies forthe control of ticks (Acari Ixodidae)rdquo Systematic and AppliedAcarology vol 3 pp 19ndash28 1998
[43] A J O Lopes J R S Nascimento-Junior C G Silva A PPrado M B Labruna and L M Costa-Junior ldquoParasitismby ixodiphagus wasps (Hymenoptera Encyrtidae) in Rhipi-cephalus sanguineus and Amblyomma ticks (Acari Ixodidae) inthree regions of Brazilrdquo Journal of Economic Entomology vol105 no 6 pp 1979ndash1981 2012
8 BioMed Research International
[44] M Bohacsova O Mediannikov M Kazimirova D Raoult andZ Sekeyova ldquoArsenophonus nasoniae and rickettsiae infectionof Ixodes ricinus due to parasitic wasp Ixodiphagus hookerirdquoPLoS ONE vol 11 no 2 Article ID e0149950 2016
[45] HOgata B La Scola S Audic et al ldquoGenome sequence ofRick-ettsia bellii illuminates the role of amoebae in gene exchangesbetween intracellular pathogensrdquo PLoS Genetics vol 2 no 5pp 733ndash744 2006
[46] S J Perlman M S Hunter and E Zchori-Fein ldquoThe emergingdiversity of Rickettsiardquo Proceedings of the Royal Society B Bio-logical Science vol 273 no 1598 pp 2097ndash2106 2006
[47] H Klompen and D Grimaldi ldquoFirst mesozoic record of aparasitiform mite A larval argasid tick in Cretaceous Amber(Acari Ixodida Argasidae)rdquoAnnals of the Entomological Societyof America vol 94 no 1 pp 10ndash15 2001
[48] D A Grimaldi M S Engel and P C Nascimbene ldquoFossilifer-ous cretaceous amber fromMyanmar (Burma) Its rediscoverybiotic diversity and paleontological significancerdquo AmericanMuseum Novitates vol 3361 pp 1ndash71 2002
[49] S Nava A A Guglielmone and A J Mangold ldquoAn overview ofsystematics and evolution of ticksrdquo Frontiers in Bioscience vol14 no 8 pp 2857ndash2877 2009
[50] A A Guglielmone D A Apanaskevich A Estrada-Pena R GRobbins T N Petney and I G Horak The Hard Ticks of theWorld (Acari Ixodida Ixodidae) vol 1 Springer Netherlandspress Dordrecht 1st edition 2014
Hindawiwwwhindawicom
International Journal of
Volume 2018
Zoology
Hindawiwwwhindawicom Volume 2018
Anatomy Research International
PeptidesInternational Journal of
Hindawiwwwhindawicom Volume 2018
Hindawiwwwhindawicom Volume 2018
Journal of Parasitology Research
GenomicsInternational Journal of
Hindawiwwwhindawicom Volume 2018
Hindawi Publishing Corporation httpwwwhindawicom Volume 2013Hindawiwwwhindawicom
The Scientific World Journal
Volume 2018
Hindawiwwwhindawicom Volume 2018
BioinformaticsAdvances in
Marine BiologyJournal of
Hindawiwwwhindawicom Volume 2018
Hindawiwwwhindawicom Volume 2018
Neuroscience Journal
Hindawiwwwhindawicom Volume 2018
BioMed Research International
Cell BiologyInternational Journal of
Hindawiwwwhindawicom Volume 2018
Hindawiwwwhindawicom Volume 2018
Biochemistry Research International
ArchaeaHindawiwwwhindawicom Volume 2018
Hindawiwwwhindawicom Volume 2018
Genetics Research International
Hindawiwwwhindawicom Volume 2018
Advances in
Virolog y Stem Cells International
Hindawiwwwhindawicom Volume 2018
Hindawiwwwhindawicom Volume 2018
Enzyme Research
Hindawiwwwhindawicom Volume 2018
International Journal of
MicrobiologyHindawiwwwhindawicom
Nucleic AcidsJournal of
Volume 2018
Submit your manuscripts atwwwhindawicom
8 BioMed Research International
[44] M Bohacsova O Mediannikov M Kazimirova D Raoult andZ Sekeyova ldquoArsenophonus nasoniae and rickettsiae infectionof Ixodes ricinus due to parasitic wasp Ixodiphagus hookerirdquoPLoS ONE vol 11 no 2 Article ID e0149950 2016
[45] HOgata B La Scola S Audic et al ldquoGenome sequence ofRick-ettsia bellii illuminates the role of amoebae in gene exchangesbetween intracellular pathogensrdquo PLoS Genetics vol 2 no 5pp 733ndash744 2006
[46] S J Perlman M S Hunter and E Zchori-Fein ldquoThe emergingdiversity of Rickettsiardquo Proceedings of the Royal Society B Bio-logical Science vol 273 no 1598 pp 2097ndash2106 2006
[47] H Klompen and D Grimaldi ldquoFirst mesozoic record of aparasitiform mite A larval argasid tick in Cretaceous Amber(Acari Ixodida Argasidae)rdquoAnnals of the Entomological Societyof America vol 94 no 1 pp 10ndash15 2001
[48] D A Grimaldi M S Engel and P C Nascimbene ldquoFossilifer-ous cretaceous amber fromMyanmar (Burma) Its rediscoverybiotic diversity and paleontological significancerdquo AmericanMuseum Novitates vol 3361 pp 1ndash71 2002
[49] S Nava A A Guglielmone and A J Mangold ldquoAn overview ofsystematics and evolution of ticksrdquo Frontiers in Bioscience vol14 no 8 pp 2857ndash2877 2009
[50] A A Guglielmone D A Apanaskevich A Estrada-Pena R GRobbins T N Petney and I G Horak The Hard Ticks of theWorld (Acari Ixodida Ixodidae) vol 1 Springer Netherlandspress Dordrecht 1st edition 2014
Hindawiwwwhindawicom
International Journal of
Volume 2018
Zoology
Hindawiwwwhindawicom Volume 2018
Anatomy Research International
PeptidesInternational Journal of
Hindawiwwwhindawicom Volume 2018
Hindawiwwwhindawicom Volume 2018
Journal of Parasitology Research
GenomicsInternational Journal of
Hindawiwwwhindawicom Volume 2018
Hindawi Publishing Corporation httpwwwhindawicom Volume 2013Hindawiwwwhindawicom
The Scientific World Journal
Volume 2018
Hindawiwwwhindawicom Volume 2018
BioinformaticsAdvances in
Marine BiologyJournal of
Hindawiwwwhindawicom Volume 2018
Hindawiwwwhindawicom Volume 2018
Neuroscience Journal
Hindawiwwwhindawicom Volume 2018
BioMed Research International
Cell BiologyInternational Journal of
Hindawiwwwhindawicom Volume 2018
Hindawiwwwhindawicom Volume 2018
Biochemistry Research International
ArchaeaHindawiwwwhindawicom Volume 2018
Hindawiwwwhindawicom Volume 2018
Genetics Research International
Hindawiwwwhindawicom Volume 2018
Advances in
Virolog y Stem Cells International
Hindawiwwwhindawicom Volume 2018
Hindawiwwwhindawicom Volume 2018
Enzyme Research
Hindawiwwwhindawicom Volume 2018
International Journal of
MicrobiologyHindawiwwwhindawicom
Nucleic AcidsJournal of
Volume 2018
Submit your manuscripts atwwwhindawicom
Hindawiwwwhindawicom
International Journal of
Volume 2018
Zoology
Hindawiwwwhindawicom Volume 2018
Anatomy Research International
PeptidesInternational Journal of
Hindawiwwwhindawicom Volume 2018
Hindawiwwwhindawicom Volume 2018
Journal of Parasitology Research
GenomicsInternational Journal of
Hindawiwwwhindawicom Volume 2018
Hindawi Publishing Corporation httpwwwhindawicom Volume 2013Hindawiwwwhindawicom
The Scientific World Journal
Volume 2018
Hindawiwwwhindawicom Volume 2018
BioinformaticsAdvances in
Marine BiologyJournal of
Hindawiwwwhindawicom Volume 2018
Hindawiwwwhindawicom Volume 2018
Neuroscience Journal
Hindawiwwwhindawicom Volume 2018
BioMed Research International
Cell BiologyInternational Journal of
Hindawiwwwhindawicom Volume 2018
Hindawiwwwhindawicom Volume 2018
Biochemistry Research International
ArchaeaHindawiwwwhindawicom Volume 2018
Hindawiwwwhindawicom Volume 2018
Genetics Research International
Hindawiwwwhindawicom Volume 2018
Advances in
Virolog y Stem Cells International
Hindawiwwwhindawicom Volume 2018
Hindawiwwwhindawicom Volume 2018
Enzyme Research
Hindawiwwwhindawicom Volume 2018
International Journal of
MicrobiologyHindawiwwwhindawicom
Nucleic AcidsJournal of
Volume 2018
Submit your manuscripts atwwwhindawicom
![SerologicalEvidenceof Rickettsia ExposureamongPatientswith ...downloads.hindawi.com/journals/ipid/2020/4905783.pdf · Rickettsia infection includes fever, eschar, and rash [6]; however,](https://static.fdocuments.in/doc/165x107/604d43e7c243f216ab359e57/serologicalevidenceof-rickettsia-exposureamongpatientswith-rickettsia-infection.jpg)
